Most represented proteins of milk exosomes.
\r\n\t
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Exosomes deliver molecular signals to recipient cells and also carry unique markers of the parental cell, which makes them a promising substrate for noninvasive diagnostics (liquid biopsy) and targeted drug delivery. The microRNA and protein contents of exosomes are of particular interest [2, 3].
\nMilk contains proteins, lipids, nucleic acids, and its complexes and is not a simple source of nutrients. Since vesicles of different size and shapes were described in milk and since milking animals can produce liters of milk per day, which is much higher than the volume of culture fluid or blood plasma, milk is a unique source of exosomes. Unfortunately, milk exosomes are currently much less studied than exosomes of blood or culture fluid.
\nMilk exosomes may be used as carriers of drugs and therapeutic nucleic acids for delivery to cells. The basis of biological functions of milk exosomes is due to their components: lipids, proteins, and nucleic acids. The prospects for the further practical use of milk exosomes in medicine and biotechnology largely depend on investigations of the fine structure and biological functions of exosomes free of various contaminating impurities.
\nA clear classification of extracellular vesicles is difficult due to their considerable variability and, particularly, overlapping sizes. The term “extracellular vesicles” was introduced to designate all the vesicles secreted to the biological fluids, and the use of the term “exosomes” requires the fulfillment of some conditions [4, 5]. The distinctive features of exosomes are the size (40–100 nm), floating density in a sucrose gradient (1.1–1.19 g/ml), a particular cup-shaped form under electron microscope examination, and specific biochemical composition.
\nExosomes isolated from human [6], cow [7], horse [8], pig [9], rat [10], and camel [11] milk have been described so far. However, it has been shown that the molecular composition of human milk exosomes largely depends on the mother’s lifestyle, lactation stage, and mother’s contact with allergens [12]. It is known that a change in the composition of proteins [13] and nucleic acids [14] of cow milk exosomes occurs during the development of inflammation of the mammary gland.
\nAccording to the international nomenclature, exosomes include vesicles of 40–100 nm in size, which are formed by invagination of the membrane of the multivesicular bodies and carry a number of specific markers, such as CD9, CD63, and CD81. Exosomes are round or cup-shaped.
\nMost investigations of exosomes confirm their presence using transmission electron microscopy (TEM) without a detailed analysis of the preparation composition. TEM is the best method of analysis of size, morphology, and integrity of exosomes, as well as of evaluating sample composition. TEM shows the presence of impurities in the preparations of exosomes, which can distort the results and lead to a false interpretation. For example, researchers describe samples of cow milk exosomes and present images that clearly show vesicles larger than 100 nm in size and other particles that do not have an outer membrane. However, when analyzing the results, this fact is not discussed [15].
\nTo confirm that the isolated vesicles are exosomes, the International Society for Extracellular Vesicles recommends identification of specific exosomal membrane proteins—tetraspanins CD9, CD63, and CD81—using Western blotting, flow cytometry, or immunoelectron microscopy [4, 16]. However, the first two methods fix all particles in a solution that have tetraspanins; therefore, there is no selectivity in analyzing membrane and non-membrane structures. The advantage of immunoelectron microscopy is the ability to detect exosomal markers directly on the surface of the vesicles.
\nWhen analyzing the literature data on exosomes obtained from the milk of human donor or other sources, one should take into account by which methods the exosome preparation was isolated and analyzed and what is the potential of these approaches in a generation of reliable data. Exosomes from different biological liquids including milk may be isolated with various methods such as centrifugation using special conditions, ultracentrifugation, ultracentrifugation in density gradients, and salting out and with other approaches [17, 18, 19]. It should be noted that these methods allow obtaining preparations only enriched with exosomes, but not pure ones. Different biological fluids including milk contain various proteins and their highly stable molecular weight associates, which can be co-isolated with various vesicles including exosomes during centrifugations. For example, it was recently shown that extract of the placenta and human milk contains very stable high-molecular-mass (~1000 kDa) multiprotein complexes, in which the size is comparable to some extent with various vesicles [20, 21]. Moreover, some free proteins and their complexes can nonspecifically or even specifically interact either with vesicle surface or their receptors and coprecipitate with the vesicles during centrifugations.
\nReliability of literature data is questioned in [22] where more than 200 exosome preparations were isolated from various sources using sequential centrifugations; this method has been used in many papers. Exosomes’ preparations contained many structures with low electron density and having no membranes; these structures were named “non-vesicles.” Using TEM, two main types of “non-vesicles” were described: of 20–40 nm (up 10–40% of all structures of the preparations) and of 40–100 nm. The morphology of the “non-vesicles” allowed referring them to lipoproteins of intermediate and low density (20–40 nm) and very low density (40–100 nm) [22]. Also, crude exosome preparations after different centrifugations without additional purification steps contained impurities of various components, including proteins and their complexes. In such types of exosome preparations, up to several hundred or thousand different contaminating co-isolating proteins and their complexes may be detected.
\nRecently our data confirmed the findings of [22]: using TEM we have shown that crude exosome preparations from human placenta and horse milk after centrifugation and ultracentrifugation (at 10,000 × g, 16,500 × g, and twice at 100,000 × g) contained many large >100 nm membrane vesicles, smaller <100 nm vesicles, and 20–100 nm non-vesicles [23]. Non-vesicles possessed medium electron density, distinct outer membrane, and spherical shape (Figure 1).
\nExosomes of human placenta after ultracentrifugation. (A) Vesicles <100 nm (black arrows), vesicles >100 nm (black squares), non-vesicles (white squares), amorphous aggregates of low electron density (white arrows), and particles associated with protein aggregates (white ovals). Individual structures in exosome preparations: Clumps of large vesicles (>100 nm) (B), vesicles <100 nm (C), non-vesicles (D, E), ring-shaped structures of ferritin (insert to A), clusters of aggregated proteins (F), and shapeless aggregations with low electron density (G). Images were obtained using TEM with negative staining. Scale bar corresponds to 100 nm.
We observed a similar situation in the case of crude exosome preparations obtained from horse milk. All the same, components were identified in such preparations except ring-shaped structures of ferritin (Figure 2).
\nExosome preparations of horse milk after different steps of purification: Ultracentrifugation (A–D) and gel filtration (E–I). (A) Aggregates of vesicles and non-vesicles, (B) exosomes and non-vesicles (oval), (C) non-vesicles, (D) macromolecular aggregates, (E) aggregates of large and small vesicles, (F) exosomes, (G) exosomes and non-vesicles, (H) exosomes labeled with anti-CD81 antibody conjugates with gold spheres, and (I) exosomes labeled with anti-CD63 antibody conjugates with gold spheres. The oval shapes denote non-vesicles; squares in (A and B)—Exosomes. Scale bar corresponds to 100 nm.
After preparations isolated from placenta [23] and horse milk [8] were enriched with exosomes by sequential centrifugation and ultracentrifugation, they were separated from co-isolating impurities using gel filtration on Sepharose 4B or Ultrogel (Figure 3A and B). The first peak corresponding to exosomes was successfully separated from contaminating proteins and other impurities of the second peak.
\nGel filtration of crude exosome preparations on Sepharose 4B (A) and Ultrogel (B). Сrude exosome preparations from human placenta (A) and horse milk (B) preparations were obtained by sequential centrifugation and ultrafiltration through filter 0.1 μm (—), absorbance at 280 nm (A280).
One can see from Figure 3 that gel filtration can noticeably differ in the A280 ratio of the first and second peaks. The first peak, corresponding to exosomes, can be significantly lower than the second peak, corresponding to coprecipitating impurities. As it was shown in [8, 23], the second peak may contain various proteins. Vesicle preparations of human placenta and horse milk obtained after gel filtration contained exosomes of various sizes (30–100 nm) but did not contain any visible amorphous protein material (Figures 1 and 2). In contrast to horse milk exosomes, preparations of human placenta exosomes even after gel filtration contain some supramolecular ring-shaped associates of ferritin (10–14 nm). One of the criteria for belonging vesicles to exosomes is the content of CD81 or CD63 on the surface [4]. Using TEM with anti-CD63 and anti-CD81 gold-labeled antibodies, we analyzed the vesicle preparation obtained with gel filtration (Figure 4C–G). Extra-purified exosomes obtained after gel filtration correspond to the exosomes in terms of morphology, size, and content of tetraspanins CD81 and CD63 on their surface (Figure 4).
\nPreparations of human placenta exosomes after filtration through 100 nm filter and gel filtration on Sepharose 4B. Vesicles (A) and ferritin ring structures (10–14 nm; inset in A). Exosomes purified by gel-filtration (B), labeled with conjugates of gold nanoparticles with monoclonal antibodies against tetraspanin CD81 (C–E) and CD63 (F–H). Transmission electron microscopy with negative contrast. Scale bar corresponds to 100 nm.
Thus, various kinds of centrifugation and ultracentrifugation are not enough for a full purification of exosome preparations from coprecipitating impurities. Also, for improved exosome purification, it is necessarily what kind of ultracentrifugation was performed and how many times it was performed. The relative number of vesicles containing CD9 and CD81 tetraspanins was estimated using flow cytometry after the first and second ultracentrifugation and after gel filtration (Figure 5). It was shown that vesicle preparations after first 100,000 × g ultracentrifugation contain only ~16% of CD9- and CD81-positive vesicles (Figure 5A and B). The second 100,000 × g ultracentrifugation led to the increase of these markers in ~fivefold (up to 80–87% for CD81 and CD9, respectively; see Figure 5C and D). Thus, the number of impurities in the preparations of exosomes not subjected to the second 100,000 × g ultracentrifugation can be approximately 4–6 times higher.
\nFlow cytometry analysis of exosome preparations after two stages of ultracentrifugation (A–D) and gel-filtration (E, F). The relative amount of vesicles containing CD9 (left) and CD81 (right) are shown.
Exosomes of different origins according to literature data may contain varying amounts of proteins. For example, crude preparations of exosomes of dendritic cells can include more than 150–200 different proteins [24, 25]. An even more improbable result was obtained when analyzing proteins of the milk’s exosomes of cows, whose preparations were isolated by centrifugation and ultracentrifugation in a sucrose gradient [26]. As a result, 2107 proteins have been identified, which include all major protein markers of exosomes previously detected.
\nWe have analyzed a possible number of proteins in extra-purified exosomes after gel filtration. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolysates after SDS-PAGE and 2D electrophoresis (Figure 6).
\n2D gel electrophoresis of horse milk exosome proteins. A mixture of five crude horse milk exosome preparations was obtained by centrifugation and ultrafiltration through 100 nm. The sample was separated by isoelectrofocusing and then by SDS-PAGE in denaturing conditions. The spots were stained with Coomassie R-250 and then cut; proteins were subjected to trypsinolysis for their identification using MALDI MS and MS/MS spectrometry.
Only 46 major and moderate protein spots were revealed on the gel after staining. Interestingly, only one of the spots corresponded to human serum albumin, lactoferrin, and lactadherin, while nine spots corresponded to different forms of beta-lactoglobulin. All other protein spots corresponded to various species of milk casein (number of spots): kappa-casein precursor (1), beta-casein (2), alpha-S1-casein (7), kappa-casein (10), and alpha-S1-casein precursor (14). Thus, the mixture of five relatively crude partially purified preparations of horse milk exosomes contains only nine different major and moderate proteins, while five of them consist of different caseins and their precursors.
\nIt could be assumed that during gel filtration, the loss of a large part of the exosomes may occur, and as a consequence, the number of proteins analyzed in these exosomes may be underestimated. However, human placenta vesicles after gel filtration (fraction of the first peak) contain ~78% of CD9- and 74% of CD81-positive vesicles (Figure 5E and F). Thus, the yield of particles after gel filtration was relatively high up to ~90%. A similar result was obtained for horse milk exosomes. Consequently, the loss of the main part of the exosomes during gel filtration does not occur.
\nAfter gel filtration, extra-purified exosomes isolated from horse milk contained only eight different major proteins, CD81, CD63, beta-lactoglobulin, and lactadherin, which were common to all preparations, and actin, butyrophilin, lactoferrin, and xanthine dehydrogenase which were found only in some of them [8]. Exosome preparations from human placenta contain only ten major proteins: CD81, CD63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, and serotransferrin [23].
\nAfter 2D electrophoresis, only 28 protein spots were found in human placenta exosomes, which corresponded to just nine different proteins and their isoforms. Overall, using 2 methods of electrophoretic analysis (1D and 2D electrophoresis), 12 proteins were identified in 4 exosome preparations: CD81, CD63, hemoglobin subunits, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, serotransferrin, human serum albumin, and immunoglobulins. Ferritin completely disappears after gel filtration and exosome treatment with proteolytic enzymes. After treatment of exosome preparations with trypsin and chymotrypsin, the protein bands corresponding to human serum albumin and immunoglobulins according to SDS-PAGE data almost wholly disappeared. Therefore, it cannot be excluded that human serum albumin and immunoglobulins form relatively stable complexes with exosome membrane proteins (i.e., tetraspanins) or interact directly with the surface of exosomes.
\nAuthors of [27, 28] discovered proteins of oxidative phosphorylation in the urinary exosomes. It is known that the formation of exosomes occurs in the cell cytoplasm. However, oxidative phosphorylation is a typical function of the mitochondria. Mitochondrial enzymes of the oxidative phosphorylation and Krebs cycle are absent in the cytoplasm. The question may arise how these enzymes could get into the cytoplasm and then to exosomes. Mechanism of such a process has not been described anywhere. These results may be explained if the authors destroyed cells and the mitochondria and then co-isolated proteins of oxidative phosphorylation (or fragments of the mitochondria) with exosomes.
\nSeveral articles describe casein in milk exosome preparations. Precursors of caseins undergo posttranslational processing in the Golgi complex. The cytoplasm of mammary gland cells never contains any casein that may get inside the multivesicular bodies and exosomes. As one can see from Figure 6, crude horse milk exosome preparations include 46 major and moderate protein spots (number of spots) including some spots of casein isoforms: kappa-casein precursor (1), beta-casein (2), alpha-S1-casein (7), kappa-casein (10), and alpha-S1-casein precursor. Casein isomers were found only in crude exosome preparations, obtained by centrifugation. All forms of caseins have disappeared after gel filtration, and they were found just in the second peak containing contaminating proteins. Thus, it is possible that hundreds to thousands of proteins described in crude milk exosome preparations, in fact, are not intrinsic components of exosomes and are co-isolating impurities.
\nIn this regard, we could note a critical review by academician Sverdlov, who believes that in the case of exosomes, there is an incorrect overestimated quantitative assessment of their internal molecular components, which, in his figurative expression, “would certainly make Amedeo Avogadro cry” [29]. We are sure that the real number of exosome proteins described previously using crude preparations of milk exosomes may be very much overestimated.
\nIt is necessary to note another potential method leading to overestimation of the protein content in exosomes. The technique identifies proteins of exosome crude preparations by bulk trypsinolysis of proteins with subsequent separation of peptides using various HPLC. For example, cow milk exosome preparations were isolated by centrifugation and ultracentrifugation [29]. Purified exosomes after trypsin treatment were subjected to reverse-phase chromatography and then fractionated on a nanoLC column connected to the tandem mass spectrometer. This approach generated near 2100 proteins detected in milk exosomes. The question is what possible errors in the estimation of protein number might be using this approach?
\nAfter SDS-PAGE all proteins and peptides with molecular mass 10 kDa and less usually leave the gel during the gel staining with Coomassie blue. Exosomal peptides and small proteins are still practically not investigated. It was shown that peptides and small proteins are easily detected using cyano-hydroxycinnamic acid as a matrix for MALDI-TOF MS and MS/MS; these substances may be directly detected even in native cells [30, 31, 32]. Other low-molecular-mass components (lipids, sugars, oligonucleotides) will appear in the MALDI spectra in these conditions only if its content is 100–1000-fold higher than of peptides. Vesicles, eluted from anti-CD81-Sepharose with 0.15 M NaCl (Figure 7), contained a mixture of peptides and its complexes.
\nAffinity chromatography of a mixture of five exosome preparations on anti-CD81-Sepharose: (—), absorbance at 280 nm (A280). Peak numbers and elution conditions are indicated in the picture.
Small proteins and peptides of the fraction, eluted from anti-CD81-Sepharose with 0.15 M NaCl, were analyzed by MALDI mass spectrometry in 2–12 kDa range. The samples were examined before and after destruction of exosomes with trifluoroacetic acid (Figure 8).
\nMALDI mass spectra of human placenta exosomes obtained with gel filtration (A) and anti-CD81-Sepharose before (B) and after (C) trifluoroacetic acid and acetonitrile treatment. The analysis was performed using 2–12 kDa range of MALDI-TOF mass spectrometer.
Similar results were obtained for horse milk exosomes before and after gel filtration and after affinity chromatography on anti-CD81-Sepharose. MALDI mass spectra demonstrate many various small proteins and peptides with molecular masses in the range 2–9 kDa. The fractions were treated with proteases. Figure 9A shows MALDI MS spectra of peptides before the protease treatment. Spectra after the incubation with trypsin (Figure 9B), chymotrypsin (Figure 9C), and proteinase K (Figure 9D) doesn’t contain any peaks >3 kDa but include peaks corresponding to the shorter products after hydrolysis. Masses of these shorter products do not coincide with the ones in Figure 9A.
\nMALDI mass spectra of <10 kDa extract of horse milk exosomes isolated on anti-CD81-Sepharose before (A) and after their treatment with trypsin (B), chymotrypsin (C), and proteinase K (D).
Exosomes of the horse milk and placenta in addition to proteins >10 kDa contain small proteins and peptides. These peptides may be analyzed with highly sensitive methods of shotgun ESI-MS/MS analysis and lead to the incorrect number of large proteins in the exosome preparations. The structure of these small proteins and peptides is not yet established; one cannot exclude that initially they may be fragments of larger proteins. In that case, the identification of large proteins from the results of the shotgun analysis of peptides will lead to an incorrect determination of many proteins.
\nSome literature data on the analysis of the various components of milk exosomes, including lipids, mRNA, microRNA, and proteins, are described below. In most of the published papers, milk vesicles were isolated using only different centrifugation, and analysis was done only on crude preparations of exosomes. According to our data, the second peaks after gel filtration of exosomes (Figure 3) contain many different milk proteins as well as RNA, lipids, and oligosaccharides. Thus, the analysis of crude preparations of exosomes without additional purification can lead to the overestimation of proteins and other biochemical components of exosomes. Therefore, the literature data on milk exosomes described below must be analyzed very gingerly and critically, taking into account the above data.
\nThere is no doubt that the biological functions of milk exosomes are due to the proteins, lipids, and nucleic acids that make up their composition. There are different estimations of the number of protein and nucleic acid molecules, which may be located in exosomes. Up to 16–20 thousands of different mRNA [33, 34] and up to 2 thousands of proteins [35] are described in cow and human exomes in various works. Prof. Sverdlov in [29] shows that no more than 1,6 thousand of mRNA molecules may fit in a single exosome, since it sounds very doubtful that such amount of different mRNA molecules may be transferred with exosomes. We believe that the papers describing thousands of proteins and nucleic acids in the preparations of exosomes may be due to the analysis of insufficiently purified preparations of exosomes, co-isolating with milk proteins and nucleic acids (of fat milk globules or other vesicular or non-vesicular particles). This assumption is well supported by the data, described in [8, 23]. As mentioned above, most papers dedicated to the study of exosomes analyze very crude preparations. Below we describe the structure and possible biological functions of major components of milk exosomes following the data given in various papers without critical analysis. However, taking into account the data provided above, it is necessary to examine the given information critically.
\nData on the protein composition of milk exosomes significantly varies between papers. There is no doubt that proteins play a crucial role in the physiology of milk exosomes; the other obvious thing is that most of the proteins, secreting in milk, cannot be a natural part of exosomes, due to the mechanism of exosome generation in the cell [8]. It was shown that the exosome proteome changes depending on the physical activity, nutrition, or various infections, suggesting that protein content of milk is an excellent biomarker [13]. Also, the data indicate that the concentration of exosomal proteins and the exosomes in milk is significantly lower in mature milk than the early stages of lactation [12].
\nDepending on the method of proteome analysis used, researchers found hundreds of proteins in milk obtained from different mammals: 100 [36], 200 [37], 400 [38], 600 [33], and even 1900 [35] or 2100 [26] proteins in exosome preparations. Such a significant difference may be due both to different levels of purification of the exosome preparations and to a substantial difference in the protein content in vesicles obtained from various sources. However, it is evident that exosomes cannot contain several thousand proteins.
\nAccording to literature data, exosomes can contain proteins that control the cytoskeleton dynamics and membrane fusion: Rab proteins (GTPase family) [39]; Alix, TSG101, and other proteins of the endosomal sorting complex [40]; and proteins that participate in the binding and transport of miRNA, target cell recognition, and fusion (tetraspanins CD9, CD63, CD81). Members of the tetraspanins family mediate the adhesion of exosomes on the surface of the recipient cell and are essential structural components of exosomal membranes [41, 42]. Also, one cannot exclude that exosomes may contain various enzymes: proteases and their activators, peroxidases, lipid kinases, and other proteins that exhibit catalytic activity [43]. Exosomes may be rich in cytoskeleton proteins (actin, tubulin, cofilin), proteins of membrane transport and heat shock (HSP60, HSP70, HSP90), and proteins involved in intracellular signal transduction like Wnt proteins, which activate the Wnt signaling pathway [44, 45]. The presence of proteins involved in the formation of vesicles (ADP-ribosylation factor) [46] and membrane fusion EHD1 (testilin) [47] confirms the endosomal origin of exosomes. The presence of integrins in milk exosomes is an essential marker of the internalization and biological activity of extracellular vesicles, which can also be used to predict their possible direction of delivery [48]. In general, exosomes include proteins that make up the endosome, plasma membrane, or cytosol, while proteins from the nucleus, mitochondria, endoplasmic reticulum, and Golgi must be absent in exosomes. These observations emphasize the specificity of the formation of these vesicles and demonstrate that exosomes are a special subcellular compartment but not random cell fragments [49].
\nThe proteins described above are common to all types of exosomes, including milk exosomes, which in addition to these proteins contain specific milk proteins. Several milk proteins were characterized in milk exosomes: caseins, lactoglobulin, lactoferrin, CD36, and polymeric immunoglobulin receptor precursor [6]. The CD36 protein in cells mediates phagocytosis and cell adhesion and binds low-density oxidized lipoproteins [50], but its role in milk exosomes requires further establishment. It is worth to say that not all the proteins described in the milk exosomes in literature may be really exosome components. For example, in highly purified preparations from horse milk, the caseins were not defined as exosomal components but co-isolated with exosomes during the ultrafiltration and ultracentrifugation and were separated from exosomal fraction by gel filtration [8]. In this regard, additional studies of milk exosomes that do not contain impurities of co-isolating proteins are required. Improvement of methods of exosome purification will make it possible to determine the protein composition of milk exosomes more accurately and to understand the role of specific proteins in their structure and functions.
\nSince the formation of the exosomal membrane occurs from the endosomal membrane, the exosomes carry similar protein markers as the cells secreting them. An example is the peripheral membrane protein MFG-E8 (lactadherin), a glycoprotein containing several domains, some of which are necessary for binding to the membrane, while some others carry integrin binding sites. Expression of MFG-E8 in the cells increases the secretion of vesicles since MFG-E8 may play a unique role in the secretion of membrane vesicles by budding or splitting the plasma membrane or by exocytosis of multivesicular bodies [51]. The expression of this protein in mammary gland increases during lactation. Human milk exosomes contain TGFb2 protein, the increased expression level of which is associated with the development of breast cancer [52].
\nCompared to cow milk fat globule membrane, the same proteins were the most abundant in cow milk exosomes: butyrophilin, xanthine oxidase, adipophilin, and lactadherin [26]. During the mastitis caused by
Flow cytometry has shown that human B-cell exosomes, unlike human milk ones, give a positive signal to tetraspanins CD40, CD54, and CD80 in addition to CD63 and CD81, which are detected in milk exosomes. On the contrary, MUC-1 protein is present in milk exosomes but absent in B-cell-derived exosomes [6].
\nIn the sediments obtained by the centrifuging of cow’s milk at 35,000 × g and 100,000 × g, CD9, CD63, and CD81 tetraspanins were found mainly in the sediment after 100,000 × g, indicating the presence of exosomes. Also, sediment obtained by 100,000 × g centrifugation contained a higher number of complement proteins C2, C6, and C7 than the 35,000 × g one, which indicates the presence of multiple types of extracellular vesicles of this sediment. Complement C8 beta chain, C1GALT1-specific chaperone 1, cartilage-associated protein, α-mannosidase 2, and procollagen-lysine 2-oxoglutarate 5-dioxygenase 3 were found only in the 100,000 × g fraction. Functional analysis of these proteins revealed three functions: galactosidase, glycosyltransferase, and peptidase activities. It cannot be excluded that proteins found in the milk exosome fraction are involved in the regulation of translation, protein maturation, and maintenance of the cellular structure. Also, analysis of human cells after fusion with milk exosomes has shown some exciting changes in expression of proteins responsible for translation (ribosomal subunits, initiation, and elongation), innate immunity, vesicular transport, and cell migration [48].
\nSome data indicate that human milk exosomes have a unique composition of proteins, distinct from other milk components. Transmembrane (CD9, CD63, CD81, lactadherin, guanine nucleotide-binding protein), cytosolic (annexins A2, A4–7, and A11, Ras-related proteins Rab, syntenin), and also intracellular proteins (endoplasmin, calnexin) are presented in human milk exosomes. The unique combination of proteins with different biological roles, cell growth, inflammation, and others indicates that milk exosomes may play a key role in the infant’s intestinal immune system. The list of the most represented exosomal proteins according to the literature data is combined in Table 1.
\nSource of milk exosomes | \nMost represented proteins | \nNumber of proteins described | \nMethod of protein analysis | \nProtein content compared in | \n
---|---|---|---|---|
Cow [26] | \nButyrophilin Xanthine oxidase Adipophilin Lactadherin | \n2107 | \nTrypsinolysis, LC-MS/MS | \nExosomes and milk fat globule membrane | \n
Human [36] | \nLactoferrin Tenascin Serum albumin β-Casein Xanthine dehydrogenase Polymeric Ig receptor | \n115 | \nTrypsinolysis, LC-MS/MS | \nDuring 12 months of lactation | \n
Cow [13] | \nButyrophilin Xanthine dehydrogenase Lactadherin Fatty acid synthase | \n2299 | \nTrypsinolysis, LC-MS/MS with iTRAQ | \nNorm and mastitis | \n
Human [35] | \nCD9, CD63, CD81 Flotilin Lactadherin Annexins G-protein subunits Ras-related proteins Rab syntenin | \n2698 | \nSDS-PAGE, trypsinolysis, LC-MS/MS | \nExtracellular vesicles and high-density complexes | \n
Horse [8] | \nβ-Lactoglobulin Lactadherin Actin Butyrophilin Lactoferrin | \n8 | \nSDS-PAGE, 2D-electrophoresis, trypsinolysis, MALDI-TOF-MS/MS | \nBefore and after gel filtration | \n
Swine [33] | \nFibronectin Thrombospondin Albumin Lactotransferrin Ceruloplasmin Complement C4 α-Glucosidase | \n571 | \nSDS-PAGE, trypsinolysis, LC-MS/MS | \n\n |
Most represented proteins of milk exosomes.
The data on the protein composition of milk exosomes gives new information on the structure and biological significance of these vesicles and reveals the potential role of exosomes in the physiology of the mammary gland. Investigation of the proteome of highly purified milk exosomes compared to milk proteome can shed light on the real protein composition of exosomes; these data may be translated to the exosomes obtained from other biological liquids. Results of milk exosome proteome analysis will possibly lead to their use in medicine as biocompatible carriers of drugs or personal therapy tools.
\nHowever, as shown above, not all proteins found in crude vesicle preparations are proper exosome proteins. At the same time, it is possible that some proteins associated with the surface of the vesicles may also play some unique role in the exosomes’ functioning. The identification of hundreds and thousands of proteins in the composition of the exosomes seems to us enormously overestimated. Also, the diversity of proteins found in exosomes raises questions about whether proteins that coprecipitated with these vesicles, as well as possible intrinsic minor proteins of exosomes, have an or have no important role in the biological functions of exosomes.
\nThe exosome membrane is enriched with specific lipids (phosphatidylcholine, cholesterol, sphingomyelin, ceramides) and has a unique protein composition that characterizes them as independent compartments [53, 54]. The minimum size of exosomes depends on the structure of the lipid bilayer, which is about 5 nm thick and has sufficient rigidity to form vesicles of 40 nm in size [55]. The lipid composition of exosomes greatly varies, due to differences in cell types, conditions of cell growth and development, as well as the use of different methods for isolating exosomes and analyzing them.
\nThe first works on the lipid composition of exosomes were carried out using a thin layer and gas-liquid chromatography. The results obtained using these methods cannot be considered fully quantitative, since the phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidic acids migrate together and are presented in the single band after separation. Similarly, sphingomyelin and ganglioside GM3 are not separated and, therefore, are taken into account together in the quantitative analysis. Today, these methods are considered obsolete for the analysis of lipids of exosomes, and most studies use modern mass spectrometry technology [56].
\nIt was found that different classes of lipids are asymmetrically distributed in the membrane of exosomes, so sphingomyelin and other sphingolipids, as well as phosphatidylcholines, are mainly located in the outer layer of the membrane, while the different classes of lipids are located in the inner layer [57]. However, the established asymmetry of the membrane bilayer can be altered by the action of particular enzymes, such as flippases, floppases, and scramblases [58, 59].
\nPhosphatidylserine in exosomes, as in the plasma membrane, is located in the inner lipid layer [57], but several studies have shown its presence also in the outer layer together with annexin 5. It is known that the presence of phosphatidylserine in the outer lipid layer activates blood cells and acts as a signal to macrophages to capture [60].
\nThe composition of certain classes of lipids in the exosomal membranes secreted by different cell types may be similar or not to the parental cells. Exosomes contain up to 2–3 times more cholesterol, sphingomyelin, glycosphingolipids, serine, and saturated fatty acids. In addition, GM3 ganglioside [56, 61], ceramides, and their derivatives [57, 62, 63] are also present in exosomes in significant amounts. In most cases, exosomes contain fewer phosphatidylcholines than their parent cells, and no noticeable differences in the content of phosphatidylserine in exosomes and parent cells were found [64].
\nThe bis(monoacylglycero)phosphates (BMP) are present in the membranes of the intraluminal vesicles of multivesicular bodies and, as stated in [57], can also be contained in the exosomes. It was shown that BMP are not transferred to exosomes but, with high probability, are included in the intraluminal vesicles of those multivesicular bodies that are associated with lysosomes. In this regard, it is believed that the primary purpose of the BMP is to promote the stability and integrity of the lysosomes. Also, it is a necessary cofactor in the process of catabolism of sphingolipids in lysosomes [65].
\nThe stiffness of exosomal membranes increases with the transition from acidic to neutral pH values, suggesting that during the secretion of the exosomes from the multivesicular bodies, some reorganization of the membrane occurs. At neutral pH, the packaging of lipids on the surface of exosomes is dense, but the transmembrane movement of lipids increases. Such a flip-flop effect (the transition of an individual molecule from one layer to another) disrupts the asymmetric distribution of lipids between the membrane layers. A uniform distribution of phosphatidylethanolamine between the two layers of the exosomal membrane [66] was shown; on the contrary, in the plasma membrane, it is located mainly in the inner lipid layer [67].
\nThe lipids of the exosomal membranes are not inert molecules but participate in the biogenesis of vesicles and affect their biological activity. Besides, exosomes carry carbohydrate groups on the outer surface; the presence of mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans was shown [68].
\nLipids form the basis of the exosome membrane; the composition of exosomal lipids significantly differs from the composition of lipids of non-vesicular structures.
\nThe above data reflect the content of lipids and oligosaccharides in the composition of mainly crude preparations of exosomes. However, as noted above, the second peak after gel filtration of exosome preparations also contains various lipids and oligosaccharides. Therefore, it cannot be excluded that part of the lipids and polysaccharides found in the vesicles will be attributed in the future to the high-molecular complexes co-isolating with exosomes during different centrifugations.
\nSeveral works show that milk exosomes contain different types of nucleic acids, including functional mRNA with a poly(A) tract at the 3′-end [33, 34, 41] and microRNA [34, 69, 70]. Currently the composition of nucleic acids in milk exosomes is described in the case of human [71, 72], cow [73, 74], porcine [33, 75], and rat [10] milk.
\nAs we have noted above, the number of individual RNA molecules in some papers may be overestimated due to various reasons. For example, it has been shown that exosomes of porcine milk contain up to 16,304 mRNA. Most of these molecules may be involved in the development of the immune system, cell proliferation, and intercellular signal transduction. Protein products of identified mRNAs might be involved in the regulation of metabolism and the growth of the piglet’s intestines [33].
\nAmong the 19,230 mRNAs found in cow milk exosomes, the most common mRNA molecules are various isoforms of the mRNA of milk and ribosomal proteins: inositol 1,4,5-triphosphate receptor type 1; α-lactalbumin; β-lactoglobulin; caseins β, κ, and α; ribosomal proteins S28, S3, P0, L32, L21, and H1 histone; and many others. Since the expression level of most of the 50 most represented transcripts in exosomes exceeds the standard in supernatants obtained by ultracentrifugation, the authors of the [34] conclude that the mRNAs are concentrated in the exosomes of milk. Unfortunately, it is difficult to agree with this statement, since the process of enrichment of exosomes with mRNA transcript molecules during their biogenesis is entirely unclear, as well as it is difficult to suggest a mechanism by which this unimaginable number of mRNA molecules can fit into an exosome with a diameter of 40–100 nm. At the same time, human and porcine milk exosomes contain little or no 18S and 28S rRNA [41, 70], which correlate with the mechanism of exosome biogenesis.
\nIt is shown that miRNA is ubiquitous in tissues and biological fluids and was previously isolated from and associated with exosomes formed from various biological fluids (serum, saliva, urine) and homogenates of body tissues, including the mammary gland [76]. Some studies have shown that the exclusion of milk exosomes and their contents, including exosomal miRNAs, from nutrition in newborns leads to impaired purine metabolism, as well as impaired spatial learning and memory in humans and mice [72, 77]. These data indicate the undesirability of feeding newborns with infant formulas since their content of microRNAs is absent or much lower than breast milk [78].
\nAnalysis of nucleic acids isolated from human milk exosomes revealed about 452 pre-miRNAs, which is approximately 32% of 1424 miRNAs described for humans. These 452 pre-miRNAs lead to the generation of 639 mature miRNAs, many of which are involved in the regulation of immune responses. At the same time, the distribution of different miRNAs in human milk exosomes is irregular; some miRNAs are represented in a million copies and others in single molecules. Ten miRNAs compose up to 62% of the total number of miRNA; the most represented miRNAs are miR-30b-5p, miR-141-3p, miR-148a-3p, miR-182-5p, miRs let-7a-5p and let-7f-5p, miR-29a-3p, miR-146b-5p, miR-182-5p, miR-200a-3p, and miR-378a-3p [79]. Interestingly miR-148a-3p may comprise up to 35% of the total number of miRNAs of human milk exosomes. miR-148a specifically controls the expression of several genes, including the TGIF2, which encodes a transcription factor, inducing the expression of various transporters and drug-metabolizing enzymes [80], and the DNMT3B gene, which encodes DNA methyltransferase [81].
\nTranscriptome of cow milk exosomes contain various miRNAs, the most common of which are bta-miR-320a-1, bta-miR-193a, bta-miR-2284x, bta-mir-181b-1, bta -miR-19b-2, bta-miR-135a-1, bta-miR-200c, bta-miR-142, bta-miR-2887-1, bta-miR-30b, bta-miR-let7i, and bta-miR-6522 [72]. It was shown that cow milk exosomes penetrate intestinal and choroidal epithelial cells [82, 83] and macrophages [34], accumulate in peripheral tissues [84, 85], and transfer miRNA to the recipient cells [86]. Analysis of exosome bioavailability between species showed that microRNA of cow milk exosomes, after oral delivery to other organisms, is protected under the low pH, RNase, and other factors of the gastrointestinal tract [78, 87, 88].
\nAnalysis of miRNA content in porcine milk exosomes revealed 366 pre-miRNAs, which can give rise to 315 mature miRNAs, and 176 of them were described in other sources. Functional analysis of porcine milk miRNA indicates their role in immune responses, and 14 of 20 of most represented miRNAs may be involved in the regulation of milk IgA production [69]. Also, it was shown that miR-148a, widely represented in the exosomes of human [71] and cow [89] milk, is also highly expressed during lactation in exosomes of porcine milk [70]. Other highly expressed miRNAs of porcine milk are miR-181 family (181a/181b/181c/181d), miR-30 family (b/c/d/e), let-7 family (a/b/d/f), and miR-98 family. Thus, miRNAs included in these families can participate in the development of the digestive tract in piglets [69].
\nNucleic acids play an essential role in biological functions of milk exosomes. The further investigations of milk exosome miRNA and mRNA variety will significantly expand the prospects of their practical use. However, when analyzing different RNA in exosomes, it should not be forgotten that exosomal preparations used in most of the papers described above were crude. Therefore, some of the detected RNA may be in the fraction with co-isolating proteins and their complexes, which may be separated from exosomes with gel filtration.
\nThe first paper describing human milk exosomes was published in 2007 [6] and dedicated to the interaction of exosomes with blood cells in cell cultures. Preparations of human milk vesicles inhibited interleukin-2, γ-interferon, and tumor necrosis factor α production by peripheral blood mononuclear cells and stimulated the increase of Foxp3+ cell proportion in vitro.
\nExosomes are natural vesicles with very promising perspectives for drug therapeutic nucleic acid delivery into the cells. One of the unresolved problems so far is the development of universal sources for isolation of preparative amounts of exosomes. The use of milk allows researchers to obtain exosome preparation several liters of milk at once that makes milk a cheap and unique source of exosomes.
\nMilk exosomes can be used for targeted drug delivery to cells. It is shown that exosomes of cow [83] and human [88] milk penetrate intestinal crypt-like cells. The possibility of milk exosomes to be used as agents for delivery of proteins, nucleic acids, and drugs makes the subject of its investigation extremely relevant. Since the components of cow milk can be used for therapy with significant limitations, since in this case the transmission of prion diseases cannot be excluded [90], analysis of exosomes isolated from other milk sources is very actual.
\nEncapsulation of curcumin in buffalo milk exosomes increased its stability in salivary, gastric, pancreatic secrets as well as in bile juice. Also, curcumin encapsulated in milk exosomes was successfully uptaken and trans-epithelial transported in Caco-2 cells [91]. Since curcumin is a hydrophobic and water-insoluble molecule, it binds to the exosomes and probably incorporates in exosomal membranes. Hydrophobic drug molecules (paclitaxel [92], doxorubicin [93], and others) can also be delivered via milk exosomes with the same mechanism. Similar results were obtained in the case of chemically synthesized siRNA [86]. Transfection of siRNA in cow milk exosomes protects it from the activity of digestive juices and increases delivery to Caco-2 cells compared to the control samples.
\nAccording to several works, milk exosomes contain antibody molecules on the surface. Transport of IgG molecules in the intestine occurs as a result of binding to neonatal Fc receptor (FcRn). Cow milk-derived exosomes were successfully delivered to the mice liver, heart, spleen, lungs, and kidneys after the oral administration [94].
\nAuspicious work shows the possibility of transfer of bovine leukemia virus proteins Env (gp51) and Gag (p24), but not viral DNA with milk exosomes obtained from infected cattle [95]. This route of viral protein delivery doesn\'t require viral infection since that may have the potential of use for immunization and/or vaccination.
\nIncubation of human milk exosomes, but not the blood plasma exosomes with monocyte-derived dendritic cells, results in DC-SIGN inhibition of HIV infection of dendritic cells and protects from HIV transfer to CD4+ T lymphocytes [96]. These results may partially explain why some breastfeed infants do not get infected from HIV-infected mothers.
\nYak milk exosomes facilitate survival of intestine cells in hypoxic conditions in vitro and have significantly higher activity than cow milk exosomes. Yak milk-derived exosomes also decrease the expression of p53, increase the expression of oxygen-sensitive prolyl hydroxylase, and decrease the expression of vascular endothelial growth factor via the hypoxia-inducible factor-α in cell cultures [97]. Human milk exosomes protect intestinal epithelium cells in vitro from oxidative stress and probably defend newborns from enterocolitis [98].
\nIt should be noted that the correct use of vesicles for medicine still requires extra-purified exosome preparation and analysis of the functioning of such exosomes. Also, it cannot be excluded that any proteins and other biologically active molecules firmly connected with the surface of vesicles can also be necessary for the manifestation of exosome biological functions. However, this issue also requires further research.
\nMilk is more than a source of nutrients and vitamins for newborn [99]; it contains different proteins and protein complexes [100] with very diverse functions. Milk is a biological liquid containing vesicles of different size and shapes. Exosomes are natural vesicles with a diameter of 40–100 nm and are found in milk obtained from human, cow, horse, camel, mice, rat, swine, and some other mammalian species. There is no doubt that milk obtained from any source contains such structures. Many biological effects are attributed to exosomes, and researchers are particularly interested in milk exosomes, since they can be used as carriers of drugs and therapeutic nucleic acids for delivery to cells. The physicochemical properties and biological functions of exosomes are primarily determined by their biochemical composition—the structure of lipids, proteins, and nucleic acids. The prospects for the further practical use of milk exosomes in medicine and biotechnology largely depend on investigations of the fine structure and biological functions of exosomes free of various contaminating impurities.
\nThis research was made possible by a grant of the Russian Scientific Foundation to Sergey Sedykh #18-74-10055 (isolation, biochemistry, morphology of milk exosomes) and by the Russian State funded budget project of ICBFM SB RAS to Georgy Nevinsky # AAAA-A17-117020210023-1 (perspectives of use).
\nThe authors declare no conflict of interest.
Fish and fishery products have become increasingly popular due to their high demand and nutritional value. According to Food and Agriculture Organization (FAO), worldwide production of finfish, mollusks (mainly bivalves), and crustaceans are 54.3, 17.7, and 9.4 million tons respectively [1]. Human consumption of fish is around 88% of total production and among them, 44% consist of live, fresh, or chilled products, and 35% consist of frozen products [2]. But, among animal-derived products, fish is considered as the most perishable commodity as it contains a high amount of water, high post mortem pH (greater than 6), non-protein-nitrogen content, free amino acids, lower content of connective tissues, and presence of an osmoregulant, trimethylamine oxide (TMAO) [3]. Spoilage or the deterioration process refers to any change in the condition of food in terms of taste, smell, appearance, or texture and becomes undesirable or unacceptable for human consumption. Generally, the process involves 3 stages; rigor mortis, autolysis, and putrefaction. Rigor mortis or the muscle stiffening will last for hours (time may vary with temperature) after their catch. Subsequently softening occurs due to enzymatic or oxidative self-digestion, and completed by microbiological processes (putrefaction) [4].
Every year, chemical and microbial deterioration alone contributes 25% of gross primary product loss (agricultural and fishery products). Besides this, there are several other factors such as harvesting season, type of species, capturing method, handling, the time lag from catch to processing, method of processing, storage temperature, etc. that also influence the rate of spoilage. During spoilage, the breakdown of various components and formation of new compounds responsible for the off- flavor, off odor, discoloration, and texture damage of the fish meat takes place [5]. Therefore, certain food additives have been added to maintain the quality, and the shelf-life of fish and fishery products. The main aim is to combat microbial contamination as well as oxidation for the extension of product’s shelf life. Generally, lipid oxidation leads to quality deterioration, and some of them can be detected by organoleptic evaluation, but microbial contamination especially pathogenic microorganisms mostly do not produce sensory deterioration, which act as a challenge for food safety. It emphasizes the importance of the application of antimicrobials in the preservation techniques [6]. In the case of fish and fishery products, preservation techniques draw more scientific attraction, since they represent internationally traded products. Even though many strategies have been developed to prevent chemical and microbial spoilage by chemical preservation, there is still a need for the use of natural preservatives, considering consumer safety. Thus, various researches and efforts have been made to invent more natural alternative solutions in the field of food preservation.
Fish and fishery products deteriorate rapidly as a consequence of various biochemical breakdowns and microbial activities on the chemical composition of meat. The spoilage involves autolysis or self-digestion of these compounds by digestive enzymes or by free radicals [7]. The major spoilage process is lipid degradation, which mainly occurs through oxidation or hydrolysis. The oxidation could be various types such as photo-oxidation, thermal oxidation, enzymatic oxidation, and auto-oxidation. It can also be accelerated by prooxidants within the body such as hemoglobin, myoglobin, cytochrome c, etc. The process involves the reaction of unsaturated fatty acids of triglycerides with atmospheric oxygen to form unstable primary products like free fatty acids (FFAs), dienes, and peroxides and secondary products like aldehydes, ketones, alcohols, hydrocarbons, volatile organic acids, trienes, epoxy compounds and carbonyls [8]. The process of lipid hydrolysis or lipolysis is breaking down of triglycerides into FFAs by the action of enzyme lipases. Accumulation of these FFAs stimulates protein denaturation, texture damage, and drip loss by the formation of protein-lipid cross-linkages [9]. Generally, protein denaturation in fish occurs mainly by the action of proteolytic enzymes in the muscle (cathepsins) and the intestinal tract (trypsins), which results in muscle solubilization and leads to undesirable texture damage. End products like amino acids, peptides, amines, H2S, ammonia, indole, etc. will be formed and will all act as a medium for microbial growth. Microbial breakdown of amino acids will lead to bitterness, souring, bad odor, sliminess, etc. of the flesh [10].
For fish and fishery products, gram-negative bacteria like
Another important spoilage mechanism is post-mortem nucleotide catabolism, resulting in ATP depletion and subsequent formation of hypoxanthine (Hx) (Figure 1). The breakdown products do not affect the safety but sensory quality undergoes some changes [17, 18]. Based on these compounds formed, the freshness can be expressed. The ratio of inosine (Ino) and Hx to total nucleotides and their catabolic derivatives will give the K value, an indicator of loss of freshness [19]. The ratio of Hx to the total of Inosine monophosphate (IMP), Ino, and Hx will give the H value, an indicator of Hx accumulation (bitterness), and its limit for human consumption has been suggested as 60% [20]. Another quality indicator is the F value. It is the ratio of IMP to the total of IMP, Ino, and Hx, and fish with F-value of 10% and higher is considered unacceptable [21]. Thus there is a huge need for the use of additives in the food industry. Application of food additives and low-temperature preservation leads to diminution of most of the spoilage process to a greater extent.
Nucleotide catabolism as a result of autolysis and putrefaction.
By definition, additives are the substances that are added to maintain or improve the safety, freshness, taste, texture, or appearance of food. Generally additives can occur in fish and fishery products during production, processing, storage, packaging, and transportation. Additives can be of two types; Synthetic or chemical, and natural additives. Some of them are listed in Table 1.
Among chemical additives, the most common and widely used chemical is sodium chloride (NaCl). Salt drying and brining is the most traditional as well as an effective method of food preservation, and several studies have been made to explore all the preservation properties of NaCl. In Nile tilapia fillets, NaCl improved the weight and minimized drip loss [34], showed weight gain in white shrimp (
Many other compounds, including phosphates, carbonates, and sulfites, are used as major seafood additives. Phosphate compounds especially, polyphosphates (PP) have been widely used in fish and fishery products as cryoprotectant [38], gel strength, and flavor enhancer [53], for providing higher cooking yield [31], improving weight, and reducing drip loss [34, 54], modifying texture, color, and reducing cooking loss [55, 56, 57], improving quality of fillet [58], minimizing drip loss in shrimp [59, 60], drip loss in sea robin (
The use of plant-derived natural compounds such as essential oils, plant extracts, hydrocolloids, phenolic compounds, etc. is very popular in seafood preservation. Their strong antimicrobial and antioxidant activities present great potential for use in the food industry [64, 65, 66].
Plant extracts and essential oils can be derived from plant petals, leaves, fruits, peels, stems, roots, and xylems and their antioxidant effects are due to volatile organic compounds, terpenoid, and phenolic components in the plant. The inhibitory effects of essential oil on gram-negative bacteria are less than that of gram-positive bacteria as their lipopolysaccharide cell wall of gram-negative bacteria blocked the invasion of hydrophobic oils into the cell membrane [67]. Using essential oils (EOs) and plant extracts to extend shelf-life and maintain the quality of fish and fishery products has been reported frequently. Some of the recent studies of their application in fish and fishery products are represented in Table 2. However strong odor and taste, high volatility, complex chemical composition, low bioavailability and stability, and factors affecting chemical compositions like plant genetic variability, extraction techniques, etc. are some limitations for the application of these phytogenic additives [99]. Like other plant-derived products, seaweed and algal extracts are emerging as a rich source of natural antioxidants, along with many nutritional values. The three important widely used hydrocolloids are; agar-agar, align, and Carrageenan. As thickening agent agar-agar is used mainly in fish paste products. Carrageenan is used to enhance the gelling property of fish mince [100, 101, 102], and organoleptic properties of mussels and squids [103]. The sodium salt of alginic acid is widely used as a stabilizer and thickener in coating films. Sodium alginate coating with rosemary extract reduced the accumulation of biogenic amines and bacterial count in Abalone (
Additive function | Categories | Examples | Application | Reference |
---|---|---|---|---|
To maintain palatability and wholesomeness (preservatives) | Antimicrobial agents | Benzoates, Sorbates, NaCl NO3−, NO2− Organic acids, EOs | Surimi/minced fish products dried, salted or cured fish Fish fillets | [22, 23] |
Antioxidants | BHA, BHT, TBHQ , PG, Ca/Na propionate, vit E, Ascorbate, Citric acid, Erythorbic acid EOs | Fish oil Fish fillets | [24, 25, 26] | |
To enhance the appeal of foods | Flavor enhancers | MSG, CaCl2, Citric acid, Disodium guanylate/ inosinate | Ready to cook or ready to eat products | [25] |
Sweeteners | Sucrose, lactose, glucose, fructose, glycerol, sorbitol. Acesulfame K, Aspartame, Sodium cyclamate, Saccarin, Sucralose, Neotam, Neohesperidine | Crab meat Fish fillets Marinades | [27, 28] | |
Colorants | Carmine, carmosine, caramel, paprika, annatto dye Iron oxides and hydroxides, Ponceau, Cochineals, Oleoresin of turmeric, TiO2, FD&C Yellow, Astaxanthin | Paste products, pre-cooked crustaceans, salmon substitutes, surimi, fish roe and smoked fish. | [28, 29] | |
Texturizing agents; (Emulsifiers/Stabilizers/ Water-binding agents) | Polysorbates, DATEM, Agar, Alginates, Carrageenan, gum, Calcium stearate, Lecithin, Yeast, Ammonium alum, | Fish and shrimp paste/mince products, Surimi | [25, 30] | |
To aid in the processing | Moisture control | CaO, Calcium stearate, STPP | Processed products | [25] |
Reduce thaw drip | Na2CO3, SHMP, STPP, TSPP | Frozen: clams, crab, fillets, lobster, shrimp and minced fish | [31] | |
Prevent cracking of glaze | Na3PO4, Na2HPO4 | Frozen products | [31] | |
Anticaking agents | Ca3(PO4)2, Na2SiO3, Ca2SiO4, MgCO3, Na4P2O7 | Paste/minced products | [25] | |
Prevent discolouration/blackspot/browning | Na2SO3 4-Hexylresorcinol, EDTA | Crustaceans | [32] | |
Packaging gases | O2, CO2, N2, Ar, O3, N2O, He, SO2, Cl | Fish and fishery products | [33] | |
Cryoprotectants | PP | Paste products, fillets, Frozen crustaceans | [28] |
Lists of additives used in fish and fishery products.
Plant derived compounds | Preservative properties | Product | Reference |
---|---|---|---|
Rosemary | Retarded microbial growth, delay chemical deterioration, sensory qualities, and extend the shelf life for 14 days during storage. | Bonito Fish Patties | [68] |
Rosemary and basil | Inhibited the formation of TVB-N and lipid oxidation products during storage | Atlantic Mackerel | [69] |
Rosemary, laurel, thyme, and sage | Antimicrobial, antioxidant properties, and also enhanced the organoleptic quality | Rainbow trout fillet | [70] |
Rosemary, cinnamon fennel and cardamom | The counts of | Carp fingers | [71] |
Green pepper | Inhibited the growth of wild type strain of | Fish-based products | [72] |
Orange leaf | Enriched with Gelatin film showed shelf-life extension of 10 days | Shrimps | [73] |
Carvacrol, bergamot and grapefruit | Improved the quality of fresh fish and extended the shelf-life up to 4 days. | Seabream | [74] |
Oregano, thyme, and star anise | Inhibited microbial growth and delaying lipid oxidation | Grass carp | [75] |
Ginger | Significant reduction in the TVBN and lipid oxidation | Cobia steaks | [76] |
Clove, cumin, and spearmint | Retarded sensory deterioration and formation of biogenic amines. | Red drum fillets | [77] |
Black cumin | Higher sensory quality, and extended shelf life | Fresh fish fillets | [78] |
limonene | Maintained spoilage bacteria at a lower level and extended the shelf-life of 15 days | Gilthead sea bream fillets | [79] |
Allyl Isothiocyanate | Extended the shelf-life by maintain specific spoilage organisms at a lower level | Gilthead sea bream | [80] |
Oregano | Activity against | Salmon | [81] |
Fennel | Coating with chitosan nanoparticles reduced the PV, TVBN, TBARS and microbial count | [82] | |
Reduced peroxide value and eliminates secondary oxidation products | Gilthead seabream fillets | [83] | |
Lemon | Lowered accumulation of histamine, and improved sensory characteristics | Salted sardines | [84] |
cinnamon bark | Inhibited | Grass carp | [85] |
Cumin seed and Wild mint leaf | Retarded microbial growth, chemical deterioration, and improved sensory characteristics | Rainbow trout fillets | [86] |
Shallot fruit and ajwain seed | Delayed lipid oxidation and microbiological spoilage | Semi-fried coated rainbow trout fillets | [87] |
Significantly delayed oxidative deterioration and maintain lower bacterial growth | Silver carp fillets | [88] | |
Colorant, preservative, and antimicrobial action | Burger and surimi | [89] | |
Potential to preserve the fresh fish during transportation | Nile tilapia | [90] | |
Mint leaf and citrus peel | Retarded the quality changes and extended the shelf life | Indian mackerel | [91] |
Inhibition to | Fresh and cold storage fish | [92] | |
Kakadu plum | Inhibited bacterial growth for 15 days at 4°C | Fish fillets | [93] |
Clove, Sage and kiwifruit peel | Antioxidant and antimicrobial potential, and extend the shelf life | Fish fingers | [94] |
Pomegranate, rosemary, and olive | Delayed the lipid oxidation, and microbial count | Fish patties | [95] |
Improved the color, rehydration and water activity | Fish oil fortified soup powder | [96] | |
Green Tea Leaves and Fenugreek Seeds | Decreased TVBN, TBARS, total bacterial count, and pH | Shrimp | [97] |
Basil leaf | Antimicrobial effect and longest shelf life | Mullet fillet | [98] |
Summary of some of the recent studies of application of essential oils, and plant extracts in fish and fishery products.
Nowadays, animal-derived products like chitosan, gelatin, and whey proteins are widely used as food additives. Chitosan is a natural polymer obtained from chitin, a component of the exoskeleton of shellfish and fungal cell walls. Gelatin is a protein derived from the raw collagen of animal body parts. Whey protein is one of the two proteins, other than casein, found in milk. The bioactive coating of food products with these compounds provides antioxidant and antimicrobial properties and can thereby increase the shelf life of the product. Direct addition of compounds into the packaging materials also provided more potent preservative action [120]. Some of the recent studies on the application of chitosan, gelatin, and whey protein as edible coatings in fish and fish product preservation are represented in Table 3. But in moist environments, edible films and coating showed relatively low stiffness and strength, thus limited their use in specific conditions. Another animal-derived product, bioactive peptide (specific protein fragments) showed antimicrobial [137], and antioxidant activities [138]. In fish paste products, the products like plasma hydrolysate, plasma protein, ovomucoid, egg albumin, egg white, etc. were added as additives for improving strength [119]. The binding effect of egg whites and hydrolyzed beef plasma proteins in surimi gels [139], gel enhancing effect of bovine plasma powder, and egg white powder in arrow tooth surimi [140] were also reported.
Fish product | Preservative action | Reference |
---|---|---|
Grass carp | Inhibited cathepsin activities and thereby retarded the proteolysis | [121] |
Olive flounder | Coating combined with clove oil improved the quality and shelf life was extended by 6 days | [122] |
White shrimp | Coating combined with pomegranate peel extract reduced the melanosis, TVBN and microbial count | [123] |
Mackerel | Coating combined with gallic acid decreased microbial growth, protein decomposition, biogenic amine formation, lipid oxidation and nucleotide breakdown and shelf life was extended | [124] |
Fish burgers | The chitosan film containing lactoperoxidase system suppress the increase of | [125] |
Mori Fish | Spoilage reduction when coated with rosemary extract on storage | [126] |
Rainbow trout fillets | Coating incorporating oregano essential oil minimized the formation of volatile bases, oxidation products, and the growth of total and psychrotrophic bacteria | [127] |
Shrimp | Fish gelatin reduced lipid oxidation and extend shelflife | [128] |
Shrimp | Coating enriched with orange leaf essential oil extend shelf life about 10 days | [73] |
Tilapia fillets | Combined with grape seed extract reduced the formation of undesirable metabolites like TMA and histidine significantly | [129] |
Golden- pomfret fillets | Inhibits myofibril degradation | [130] |
White shrimp | Decreased the total and psychrotrophic bacteria and increased the shelf-life | [131] |
Salmon fillet | Incorporated with garlic and lime extracts extended the shelf life by antibacterial and antioxidant activity | [132] |
Minced trout fillet | Incorporated with grape seed extract | [133] |
Pike-Perch | Coating with lactoperoxidase system and α-tocopherol extended the shelflife significantly | [134] |
Rainbow trout fillet | Reduced microbial growth, and TVB-N and TBA | [135] |
Rainbow trout fillet | Inhibited | [136] |
Some of the recent studies on the application of chitosan, gelatin, and whey protein as edible coatings in fish and fish product preservation.
Bacteriocin, a major bacterial-derived bio preservative (mostly from
As a perishable food commodity, most of the world’s supply of fish and fishery products are lost through chemical and microbial spoilage than other reasons like improper storage, handling and processing damage. Thus, the increasing demand for good quality fish products has intensified the search for applications of additives in preservation strategies. It is well known that none of the additives offer complete protection against spoilage, but can improve the quality of fish as well as shelf life to a greater extent. By considering the potential health hazards associated with chemicals as well as consumer preference, application of natural products from cheap and underutilized resources enabling food safety holds promise.
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All published Book Chapters are licensed under a Creative Commons Attribution 3.0 Unported License. Monographs are licensed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) license granted to all others. Our Copyright Policy aims to guarantee that original material is published while at the same time giving significant freedom to our Authors. IntechOpen upholds a flexible Copyright Policy meaning that there is no copyright transfer to the publisher and Authors hold exclusive copyright to their work.
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\n\nAll scientific works are subject to Peer Review prior to publishing. IntechOpen is a member of the Committee on Publication Ethics (COPE) and all participating referees and Academic Editors are expected to review submitted scientific works in line with the COPE Ethical Guidelines for Peer Reviewers where applicable.
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On September, 29th 2006 he has won a post PhD fellowship from the university of Bologna (from October 2006 to October 2008), at the competitive examination he was ranked first in the industrial engineering area. He extensively served as referee for several international journals. He is author/coauthor of more than 100 research papers. He has been involved in some projects supported by MURST and European Community. His research interests include pattern recognition, bioinformatics, and biometric systems (fingerprint classification and recognition, signature verification, face recognition).",institutionString:null,institution:null},{id:"496",title:"Dr.",name:"Carlos",middleName:null,surname:"Leon",slug:"carlos-leon",fullName:"Carlos Leon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Seville",country:{name:"Spain"}}},{id:"512",title:"Dr.",name:"Dayang",middleName:null,surname:"Jawawi",slug:"dayang-jawawi",fullName:"Dayang Jawawi",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Technology Malaysia",country:{name:"Malaysia"}}},{id:"528",title:"Dr.",name:"Kresimir",middleName:null,surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/528/images/system/528.jpg",biography:"K. Delac received his B.Sc.E.E. degree in 2003 and is currentlypursuing a Ph.D. degree at the University of Zagreb, Faculty of Electrical Engineering andComputing. His current research interests are digital image analysis, pattern recognition andbiometrics.",institutionString:null,institution:{name:"University of Zagreb",country:{name:"Croatia"}}},{id:"557",title:"Dr.",name:"Andon",middleName:"Venelinov",surname:"Topalov",slug:"andon-topalov",fullName:"Andon Topalov",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/557/images/1927_n.jpg",biography:"Dr. Andon V. Topalov received the MSc degree in Control Engineering from the Faculty of Information Systems, Technologies, and Automation at Moscow State University of Civil Engineering (MGGU) in 1979. He then received his PhD degree in Control Engineering from the Department of Automation and Remote Control at Moscow State Mining University (MGSU), Moscow, in 1984. From 1985 to 1986, he was a Research Fellow in the Research Institute for Electronic Equipment, ZZU AD, Plovdiv, Bulgaria. In 1986, he joined the Department of Control Systems, Technical University of Sofia at the Plovdiv campus, where he is presently a Full Professor. He has held long-term visiting Professor/Scholar positions at various institutions in South Korea, Turkey, Mexico, Greece, Belgium, UK, and Germany. And he has coauthored one book and authored or coauthored more than 80 research papers in conference proceedings and journals. 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Aalborg University has Two Satellite Campuses, one in Copenhagen (Aalborg University Copenhagen) and the other in Esbjerg (Aalborg University Esbjerg).\n· He is a member of prestigious IEEE (Institute of Electrical and Electronics Engineers), and IAENG (International Association of Engineers) organizations. \n· He is the chief Editor of the Journal of Software Engineering.\n· He is the member of the Editorial Board of International Journal of Computer Science and Software Technology (IJCSST) and International Journal of Computer Engineering and Information Technology. \n· He is also the Editor of Communication in Computer and Information Science CCIS-20 by Springer.\n· Reviewer For Many Conferences\nHe is the lead person in making collaboration agreements between Aalborg University and many universities of Pakistan, for which the MOU’s (Memorandum of Understanding) have been signed.\nProfessor Akbar is working in Academia since 1990, he started his career as a Lab demonstrator/TA at the University of Sussex. After finishing his P. hD degree in 1992, he served in the Industry as a Scientific Officer and continued his academic career as a visiting scholar for a number of educational institutions. In 1996 he joined National University of Science & Technology Pakistan (NUST) as an Associate Professor; NUST is one of the top few universities in Pakistan. In 1999 he joined an International Company Lineo Inc, Canada as Manager Compiler Group, where he headed the group for developing Compiler Tool Chain and Porting of Operating Systems for the BLACKfin processor. The processor development was a joint venture by Intel and Analog Devices. In 2002 Lineo Inc., was taken over by another company, so he joined Aalborg University Denmark as an Assistant Professor.\nProfessor Akbar has truly a multi-disciplined career and he continued his legacy and making progress in many areas of his interests both in teaching and research. He has contributed in stochastic estimation of control area especially, in the Multiple Target Tracking and Interactive Multiple Model (IMM) research, Ball & Beam Control Problem, Robotics, Levitation Control. He has contributed in developing Algorithms for Fingerprint Matching, Computer Vision and Face Recognition. He has been supervising Pattern Recognition, Formal Languages and Distributed Processing projects for several years. He has reviewed many books on Management, Computer Science. Currently, he is an active and permanent reviewer for many international conferences and symposia and the program committee member for many international conferences.\nIn teaching he has taught the core computer science subjects like, Digital Design, Real Time Embedded System Programming, Operating Systems, Software Engineering, Data Structures, Databases, Compiler Construction. 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It is, therefore, worthwhile that researchers in the African continent and the world over should keep on working on identifying biomolecules with potential in cancer management.",book:{id:"5767",slug:"natural-products-and-cancer-drug-discovery",title:"Natural Products and Cancer Drug Discovery",fullTitle:"Natural Products and Cancer Drug Discovery"},signatures:"Newman Osafo, Yaw Duah Boakye, Christian Agyare, Samuel\nObeng, Judith Edem Foli and Prince Amankwaah Baffour Minkah",authors:[{id:"182058",title:"Dr.",name:"Christian",middleName:null,surname:"Agyare",slug:"christian-agyare",fullName:"Christian Agyare"},{id:"186987",title:"Dr.",name:"Yaw Duah",middleName:null,surname:"Boakye",slug:"yaw-duah-boakye",fullName:"Yaw Duah Boakye"},{id:"196452",title:"Dr.",name:"Newman",middleName:null,surname:"Osafo",slug:"newman-osafo",fullName:"Newman Osafo"},{id:"201381",title:"Ms.",name:"Judith",middleName:null,surname:"Edem Foli",slug:"judith-edem-foli",fullName:"Judith Edem Foli"},{id:"201382",title:"Mr.",name:"Prince",middleName:"Amankwah Baffour",surname:"Minkah",slug:"prince-minkah",fullName:"Prince Minkah"},{id:"204731",title:"Mr.",name:"Samuel",middleName:null,surname:"Obeng",slug:"samuel-obeng",fullName:"Samuel Obeng"}]},{id:"53856",title:"Early-Stage Progression of Breast Cancer",slug:"early-stage-progression-of-breast-cancer",totalDownloads:1713,totalCrossrefCites:4,totalDimensionsCites:4,abstract:"Breast cancer can be defined as a group of diseases with heterogeneous origins, molecular profiles and behaviors characterized by uncontrolled proliferation of cells within the mammary tissue. Around one in eight women in the US will develop breast cancer in their lifetime, making it the second most frequently diagnosed cancer behind skin cancer [1]. In 2015, an estimated 231,840 cases of invasive carcinoma were diagnosed, and over 40,000 deaths were caused by breast cancer which accounts for almost 7% of all cancer mortality each year. In 2015, 60,290 cases of in situ breast cancer were diagnosed, representing over 14% of all new cancer cases among women and men. The steep increase in diagnosis of early‐stage breast cancer over the past 10 years is believed to be a result of more frequent mammography. However, since over half of these in situ lesions will not progress to invasive breast cancer, controversies have arisen about approaches to treatment and prevention of progression of early‐stage in situ breast cancer. Understanding the mechanisms of transition of normal breast to in situ pre‐neoplastic lesions and invasive breast cancer is currently a major focus of breast cancer research with implications for preventive and clinical management of breast cancer. In this review, we give an overview of current knowledge on the molecular and pathological changes that occur during early‐stage progression of breast cancer and describe some of the current models that are used to study this process.",book:{id:"5431",slug:"breast-cancer-from-biology-to-medicine",title:"Breast Cancer",fullTitle:"Breast Cancer - From Biology to Medicine"},signatures:"William Kietzman, Anna T. Riegel and Virginie Ory",authors:[{id:"190578",title:"Prof.",name:"Anna",middleName:null,surname:"Riegel",slug:"anna-riegel",fullName:"Anna Riegel"},{id:"190580",title:"Dr.",name:"Virginie",middleName:null,surname:"Ory",slug:"virginie-ory",fullName:"Virginie Ory"},{id:"190583",title:"MSc.",name:"William",middleName:null,surname:"Kietzman",slug:"william-kietzman",fullName:"William Kietzman"}]}],onlineFirstChaptersFilter:{topicId:"190",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:11,numberOfPublishedChapters:91,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:108,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:33,numberOfPublishedChapters:333,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:14,numberOfPublishedChapters:145,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:11,numberOfPublishedChapters:144,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!0},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:126,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:113,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:23,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:13,numberOfOpenTopics:1,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!0},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:5,numberOfUpcomingTopics:0,issn:"2753-6580",doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}},{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}}]},series:{item:{id:"11",title:"Biochemistry",doi:"10.5772/intechopen.72877",issn:"2632-0983",scope:"Biochemistry, the study of chemical transformations occurring within living organisms, impacts all areas of life sciences, from molecular crystallography and genetics to ecology, medicine, and population biology. Biochemistry examines macromolecules - proteins, nucleic acids, carbohydrates, and lipids – and their building blocks, structures, functions, and interactions. Much of biochemistry is devoted to enzymes, proteins that catalyze chemical reactions, enzyme structures, mechanisms of action and their roles within cells. Biochemistry also studies small signaling molecules, coenzymes, inhibitors, vitamins, and hormones, which play roles in life processes. Biochemical experimentation, besides coopting classical chemistry methods, e.g., chromatography, adopted new techniques, e.g., X-ray diffraction, electron microscopy, NMR, radioisotopes, and developed sophisticated microbial genetic tools, e.g., auxotroph mutants and their revertants, fermentation, etc. More recently, biochemistry embraced the ‘big data’ omics systems. Initial biochemical studies have been exclusively analytic: dissecting, purifying, and examining individual components of a biological system; in the apt words of Efraim Racker (1913 –1991), “Don’t waste clean thinking on dirty enzymes.” Today, however, biochemistry is becoming more agglomerative and comprehensive, setting out to integrate and describe entirely particular biological systems. The ‘big data’ metabolomics can define the complement of small molecules, e.g., in a soil or biofilm sample; proteomics can distinguish all the comprising proteins, e.g., serum; metagenomics can identify all the genes in a complex environment, e.g., the bovine rumen. 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Dr. Blumenberg’s research is focused on the epidermis, expression of keratin genes, transcription profiling, keratinocyte differentiation, inflammatory diseases and cancers, and most recently the effects of the microbiome on the skin. He has published more than 100 peer-reviewed research articles and graduated numerous Ph.D. and postdoctoral students.",institutionString:null,institution:{name:"New York University Langone Medical Center",institutionURL:null,country:{name:"United States of America"}}},editorTwo:null,editorThree:null},subseries:{paginationCount:4,paginationItems:[{id:"14",title:"Cell and Molecular Biology",coverUrl:"https://cdn.intechopen.com/series_topics/covers/14.jpg",isOpenForSubmission:!0,editor:{id:"165627",title:"Dr.",name:"Rosa María",middleName:null,surname:"Martínez-Espinosa",slug:"rosa-maria-martinez-espinosa",fullName:"Rosa María Martínez-Espinosa",profilePictureURL:"https://mts.intechopen.com/storage/users/165627/images/system/165627.jpeg",biography:"Rosa María Martínez-Espinosa is a Full Professor of Biochemistry and Molecular Biology at the University of Alicante, Spain, and has been the vice president of International Relations and Development Cooperation at this university since 2010. She created the research group in applied biochemistry in 2017 (https://web.ua.es/en/appbiochem/), and from 1999 to the present has made more than 200 contributions to Spanish and international conferences. Furthermore, she has around seventy-five scientific publications in indexed journals, eighty book chapters, and one patent to her credit. Her research work focuses on microbial metabolism (particularly on extremophile microorganisms), purification and characterization of enzymes with potential industrial and biotechnological applications, protocol optimization for genetically manipulating microorganisms, gene regulation characterization, carotenoid (pigment) production, and design and development of contaminated water and soil bioremediation processes by means of microorganisms. This research has received competitive public grants from the European Commission, the Spanish Ministry of Economy and Competitiveness, the Valencia Region Government, and the University of Alicante.",institutionString:"University of Alicante",institution:{name:"University of Alicante",institutionURL:null,country:{name:"Spain"}}},editorTwo:null,editorThree:null},{id:"15",title:"Chemical Biology",coverUrl:"https://cdn.intechopen.com/series_topics/covers/15.jpg",isOpenForSubmission:!0,editor:{id:"441442",title:"Dr.",name:"Şükrü",middleName:null,surname:"Beydemir",slug:"sukru-beydemir",fullName:"Şükrü Beydemir",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00003GsUoIQAV/Profile_Picture_1634557147521",biography:"Dr. Şükrü Beydemir obtained a BSc in Chemistry in 1995 from Yüzüncü Yıl University, MSc in Biochemistry in 1998, and PhD in Biochemistry in 2002 from Atatürk University, Turkey. He performed post-doctoral studies at Max-Planck Institute, Germany, and University of Florence, Italy in addition to making several scientific visits abroad. He currently works as a Full Professor of Biochemistry in the Faculty of Pharmacy, Anadolu University, Turkey. Dr. Beydemir has published over a hundred scientific papers spanning protein biochemistry, enzymology and medicinal chemistry, reviews, book chapters and presented several conferences to scientists worldwide. He has received numerous publication awards from various international scientific councils. He serves in the Editorial Board of several international journals. Dr. Beydemir is also Rector of Bilecik Şeyh Edebali University, Turkey.",institutionString:null,institution:{name:"Anadolu University",institutionURL:null,country:{name:"Turkey"}}},editorTwo:{id:"13652",title:"Prof.",name:"Deniz",middleName:null,surname:"Ekinci",slug:"deniz-ekinci",fullName:"Deniz Ekinci",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002aYLT1QAO/Profile_Picture_1634557223079",biography:"Dr. Deniz Ekinci obtained a BSc in Chemistry in 2004, MSc in Biochemistry in 2006, and PhD in Biochemistry in 2009 from Atatürk University, Turkey. He studied at Stetson University, USA, in 2007-2008 and at the Max Planck Institute of Molecular Cell Biology and Genetics, Germany, in 2009-2010. Dr. Ekinci currently works as a Full Professor of Biochemistry in the Faculty of Agriculture and is the Head of the Enzyme and Microbial Biotechnology Division, Ondokuz Mayıs University, Turkey. He is a member of the Turkish Biochemical Society, American Chemical Society, and German Genetics society. Dr. Ekinci published around ninety scientific papers, reviews and book chapters, and presented several conferences to scientists. He has received numerous publication awards from several scientific councils. Dr. Ekinci serves as the Editor in Chief of four international books and is involved in the Editorial Board of several international journals.",institutionString:null,institution:{name:"Ondokuz Mayıs University",institutionURL:null,country:{name:"Turkey"}}},editorThree:null},{id:"17",title:"Metabolism",coverUrl:"https://cdn.intechopen.com/series_topics/covers/17.jpg",isOpenForSubmission:!0,editor:{id:"138626",title:"Dr.",name:"Yannis",middleName:null,surname:"Karamanos",slug:"yannis-karamanos",fullName:"Yannis Karamanos",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002g6Jv2QAE/Profile_Picture_1629356660984",biography:"Yannis Karamanos, born in Greece in 1953, completed his pre-graduate studies at the Université Pierre et Marie Curie, Paris, then his Masters and Doctoral degree at the Université de Lille (1983). He was associate professor at the University of Limoges (1987) before becoming full professor of biochemistry at the Université d’Artois (1996). He worked on the structure-function relationships of glycoconjugates and his main project was the investigations on the biological roles of the de-N-glycosylation enzymes (Endo-N-acetyl-β-D-glucosaminidase and peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase). From 2002 he contributes to the understanding of the Blood-brain barrier functioning using proteomics approaches. He has published more than 70 papers. His teaching areas are energy metabolism and regulation, integration and organ specialization and metabolic adaptation.",institutionString:null,institution:{name:"Artois University",institutionURL:null,country:{name:"France"}}},editorTwo:null,editorThree:null},{id:"18",title:"Proteomics",coverUrl:"https://cdn.intechopen.com/series_topics/covers/18.jpg",isOpenForSubmission:!0,editor:{id:"200689",title:"Prof.",name:"Paolo",middleName:null,surname:"Iadarola",slug:"paolo-iadarola",fullName:"Paolo Iadarola",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bSCl8QAG/Profile_Picture_1623568118342",biography:"Paolo Iadarola graduated with a degree in Chemistry from the University of Pavia (Italy) in July 1972. He then worked as an Assistant Professor at the Faculty of Science of the same University until 1984. In 1985, Prof. Iadarola became Associate Professor at the Department of Biology and Biotechnologies of the University of Pavia and retired in October 2017. Since then, he has been working as an Adjunct Professor in the same Department at the University of Pavia. His research activity during the first years was primarily focused on the purification and structural characterization of enzymes from animal and plant sources. During this period, Prof. Iadarola familiarized himself with the conventional techniques used in column chromatography, spectrophotometry, manual Edman degradation, and electrophoresis). Since 1995, he has been working on: i) the determination in biological fluids (serum, urine, bronchoalveolar lavage, sputum) of proteolytic activities involved in the degradation processes of connective tissue matrix, and ii) on the identification of biological markers of lung diseases. In this context, he has developed and validated new methodologies (e.g., Capillary Electrophoresis coupled to Laser-Induced Fluorescence, CE-LIF) whose application enabled him to determine both the amounts of biochemical markers (Desmosines) in urine/serum of patients affected by Chronic Obstructive Pulmonary Disease (COPD) and the activity of proteolytic enzymes (Human Neutrophil Elastase, Cathepsin G, Pseudomonas aeruginosa elastase) in sputa of these patients. More recently, Prof. Iadarola was involved in developing techniques such as two-dimensional electrophoresis coupled to liquid chromatography/mass spectrometry (2DE-LC/MS) for the proteomic analysis of biological fluids aimed at the identification of potential biomarkers of different lung diseases. He is the author of about 150 publications (According to Scopus: H-Index: 23; Total citations: 1568- According to WOS: H-Index: 20; Total Citations: 1296) of peer-reviewed international journals. He is a Consultant Reviewer for several journals, including the Journal of Chromatography A, Journal of Chromatography B, Plos ONE, Proteomes, International Journal of Molecular Science, Biotech, Electrophoresis, and others. He is also Associate Editor of Biotech.",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorTwo:{id:"201414",title:"Dr.",name:"Simona",middleName:null,surname:"Viglio",slug:"simona-viglio",fullName:"Simona Viglio",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRKDHQA4/Profile_Picture_1630402531487",biography:"Simona Viglio is an Associate Professor of Biochemistry at the Department of Molecular Medicine at the University of Pavia. She has been working since 1995 on the determination of proteolytic enzymes involved in the degradation process of connective tissue matrix and on the identification of biological markers of lung diseases. She gained considerable experience in developing and validating new methodologies whose applications allowed her to determine both the amount of biomarkers (Desmosine and Isodesmosine) in the urine of patients affected by COPD, and the activity of proteolytic enzymes (HNE, Cathepsin G, Pseudomonas aeruginosa elastase) in the sputa of these patients. Simona Viglio was also involved in research dealing with the supplementation of amino acids in patients with brain injury and chronic heart failure. She is presently engaged in the development of 2-DE and LC-MS techniques for the study of proteomics in biological fluids. The aim of this research is the identification of potential biomarkers of lung diseases. She is an author of about 90 publications (According to Scopus: H-Index: 23; According to WOS: H-Index: 20) on peer-reviewed journals, a member of the “Società Italiana di Biochimica e Biologia Molecolare,“ and a Consultant Reviewer for International Journal of Molecular Science, Journal of Chromatography A, COPD, Plos ONE and Nutritional Neuroscience.",institutionString:null,institution:{name:"University of Pavia",institutionURL:null,country:{name:"Italy"}}},editorThree:null}]},overviewPageOFChapters:{paginationCount:45,paginationItems:[{id:"83122",title:"New Perspectives on the Application of Chito-Oligosaccharides Derived from Chitin and Chitosan: A Review",doi:"10.5772/intechopen.106501",signatures:"Paul Edgardo Regalado-Infante, Norma Gabriela Rojas-Avelizapa, Rosalía Núñez-Pastrana, Daniel Tapia-Maruri, Andrea Margarita Rivas-Castillo, Régulo Carlos Llarena-Hernández and Luz Irene Rojas-Avelizapa",slug:"new-perspectives-on-the-application-of-chito-oligosaccharides-derived-from-chitin-and-chitosan-a-rev",totalDownloads:1,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Chitin-Chitosan - Isolation, Properties, and Applications",coverURL:"https://cdn.intechopen.com/books/images_new/11670.jpg",subseries:{id:"15",title:"Chemical Biology"}}},{id:"83015",title:"Acute Changes in Lipoprotein-Associated Oxidative Stress",doi:"10.5772/intechopen.106489",signatures:"Ngoc-Anh Le",slug:"acute-changes-in-lipoprotein-associated-oxidative-stress",totalDownloads:6,totalCrossrefCites:0,totalDimensionsCites:0,authors:[{name:"Anh",surname:"Le"}],book:{title:"Importance of Oxidative Stress and Antioxidant System in Health and Disease",coverURL:"https://cdn.intechopen.com/books/images_new/11671.jpg",subseries:{id:"15",title:"Chemical Biology"}}},{id:"83041",title:"Responses of Endoplasmic Reticulum to Plant Stress",doi:"10.5772/intechopen.106590",signatures:"Vishwa Jyoti Baruah, Bhaswati Sarmah, Manny Saluja and Elizabeth H. Mahood",slug:"responses-of-endoplasmic-reticulum-to-plant-stress",totalDownloads:5,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Updates on Endoplasmic Reticulum",coverURL:"https://cdn.intechopen.com/books/images_new/11674.jpg",subseries:{id:"14",title:"Cell and Molecular Biology"}}},{id:"82914",title:"Glance on the Critical Role of IL-23 Receptor Gene Variations in Inflammation-Induced Carcinogenesis",doi:"10.5772/intechopen.105049",signatures:"Mohammed El-Gedamy",slug:"glance-on-the-critical-role-of-il-23-receptor-gene-variations-in-inflammation-induced-carcinogenesis",totalDownloads:16,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Chemokines Updates",coverURL:"https://cdn.intechopen.com/books/images_new/11672.jpg",subseries:{id:"18",title:"Proteomics"}}}]},overviewPagePublishedBooks:{paginationCount:33,paginationItems:[{type:"book",id:"7006",title:"Biochemistry and Health Benefits of Fatty Acids",subtitle:null,coverURL:"https://cdn.intechopen.com/books/images_new/7006.jpg",slug:"biochemistry-and-health-benefits-of-fatty-acids",publishedDate:"December 19th 2018",editedByType:"Edited by",bookSignature:"Viduranga Waisundara",hash:"c93a00abd68b5eba67e5e719f67fd20b",volumeInSeries:1,fullTitle:"Biochemistry and Health Benefits of Fatty Acids",editors:[{id:"194281",title:"Dr.",name:"Viduranga Y.",middleName:null,surname:"Waisundara",slug:"viduranga-y.-waisundara",fullName:"Viduranga Y. Waisundara",profilePictureURL:"https://mts.intechopen.com/storage/users/194281/images/system/194281.jpg",biography:"Dr. Viduranga Waisundara obtained her Ph.D. in Food Science\nand Technology from the Department of Chemistry, National\nUniversity of Singapore, in 2010. She was a lecturer at Temasek Polytechnic, Singapore from July 2009 to March 2013.\nShe relocated to her motherland of Sri Lanka and spearheaded the Functional Food Product Development Project at the\nNational Institute of Fundamental Studies from April 2013 to\nOctober 2016. She was a senior lecturer on a temporary basis at the Department of\nFood Technology, Faculty of Technology, Rajarata University of Sri Lanka. She is\ncurrently Deputy Principal of the Australian College of Business and Technology –\nKandy Campus, Sri Lanka. She is also the Global Harmonization Initiative (GHI)",institutionString:"Australian College of Business & Technology",institution:{name:"Kobe College",institutionURL:null,country:{name:"Japan"}}}]},{type:"book",id:"6820",title:"Keratin",subtitle:null,coverURL:"https://cdn.intechopen.com/books/images_new/6820.jpg",slug:"keratin",publishedDate:"December 19th 2018",editedByType:"Edited by",bookSignature:"Miroslav Blumenberg",hash:"6def75cd4b6b5324a02b6dc0359896d0",volumeInSeries:2,fullTitle:"Keratin",editors:[{id:"31610",title:"Dr.",name:"Miroslav",middleName:null,surname:"Blumenberg",slug:"miroslav-blumenberg",fullName:"Miroslav Blumenberg",profilePictureURL:"https://mts.intechopen.com/storage/users/31610/images/system/31610.jpg",biography:"Miroslav Blumenberg, Ph.D., was born in Subotica and received his BSc in Belgrade, Yugoslavia. He completed his Ph.D. at MIT in Organic Chemistry; he followed up his Ph.D. with two postdoctoral study periods at Stanford University. Since 1983, he has been a faculty member of the RO Perelman Department of Dermatology, NYU School of Medicine, where he is codirector of a training grant in cutaneous biology. Dr. Blumenberg’s research is focused on the epidermis, expression of keratin genes, transcription profiling, keratinocyte differentiation, inflammatory diseases and cancers, and most recently the effects of the microbiome on the skin. 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Dr. Gaiceanu is a member of the National Council for Attesting Titles, Diplomas and Certificates, an expert of the Executive Agency for Higher Education, Research Funding, and a member of the Senate of the Dunarea de Jos University of Galati. He has been the head of the Integrated Energy Conversion Systems and Advanced Control of Complex Processes Research Center, Romania, since 2016. He has conducted several projects in power converter systems for electrical drives, power quality, PEM and SOFC fuel cell power converters for utilities, electric vehicles, and marine applications with the Department of Regulation and Control, SIEI S.pA. (2002–2004) and the Polytechnic University of Turin, Italy (2002–2004, 2006–2007). He is a member of the Institute of Electrical and Electronics Engineers (IEEE) and cofounder-member of the IEEE Power Electronics Romanian Chapter. He is a guest editor at Energies and an academic book editor for IntechOpen. He is also a member of the editorial boards of the Journal of Electrical Engineering, Electronics, Control and Computer Science and Sustainability. Dr. Gaiceanu has been General Chairman of the IEEE International Symposium on Electrical and Electronics Engineering in the last six editions.",institutionString:'"Dunarea de Jos" University of Galati',institution:{name:'"Dunarea de Jos" University of Galati',country:{name:"Romania"}}},{id:"4519",title:"Prof.",name:"Jaydip",middleName:null,surname:"Sen",slug:"jaydip-sen",fullName:"Jaydip Sen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/4519/images/system/4519.jpeg",biography:"Jaydip Sen is associated with Praxis Business School, Kolkata, India, as a professor in the Department of Data Science. His research areas include security and privacy issues in computing and communication, intrusion detection systems, machine learning, deep learning, and artificial intelligence in the financial domain. He has more than 200 publications in reputed international journals, refereed conference proceedings, and 20 book chapters in books published by internationally renowned publishing houses, such as Springer, CRC press, IGI Global, etc. Currently, he is serving on the editorial board of the prestigious journal Frontiers in Communications and Networks and in the technical program committees of a number of high-ranked international conferences organized by the IEEE, USA, and the ACM, USA. He has been listed among the top 2% of scientists in the world for the last three consecutive years, 2019 to 2021 as per studies conducted by the Stanford University, USA.",institutionString:"Praxis Business School",institution:null},{id:"320071",title:"Dr.",name:"Sidra",middleName:null,surname:"Mehtab",slug:"sidra-mehtab",fullName:"Sidra Mehtab",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00002v6KHoQAM/Profile_Picture_1584512086360",biography:"Sidra Mehtab has completed her BS with honors in Physics from Calcutta University, India in 2018. She has done MS in Data Science and Analytics from Maulana Abul Kalam Azad University of Technology (MAKAUT), Kolkata, India in 2020. Her research areas include Econometrics, Time Series Analysis, Machine Learning, Deep Learning, Artificial Intelligence, and Computer and Network Security with a particular focus on Cyber Security Analytics. Ms. Mehtab has published seven papers in international conferences and one of her papers has been accepted for publication in a reputable international journal. She has won the best paper awards in two prestigious international conferences – BAICONF 2019, and ICADCML 2021, organized in the Indian Institute of Management, Bangalore, India in December 2019, and SOA University, Bhubaneswar, India in January 2021. Besides, Ms. Mehtab has also published two book chapters in two books. Seven of her book chapters will be published in a volume shortly in 2021 by Cambridge Scholars’ Press, UK. Currently, she is working as the joint editor of two edited volumes on Time Series Analysis and Forecasting to be published in the first half of 2021 by an international house. Currently, she is working as a Data Scientist with an MNC in Delhi, India.",institutionString:"NSHM College of Management and Technology",institution:{name:"Association for Computing Machinery",country:{name:"United States of America"}}},{id:"226240",title:"Dr.",name:"Andri Irfan",middleName:null,surname:"Rifai",slug:"andri-irfan-rifai",fullName:"Andri Irfan Rifai",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/226240/images/7412_n.jpg",biography:"Andri IRFAN is a Senior Lecturer of Civil Engineering and Planning. He completed the PhD at the Universitas Indonesia & Universidade do Minho with Sandwich Program Scholarship from the Directorate General of Higher Education and LPDP scholarship. He has been teaching for more than 19 years and much active to applied his knowledge in the project construction in Indonesia. His research interest ranges from pavement management system to advanced data mining techniques for transportation engineering. He has published more than 50 papers in journals and 2 books.",institutionString:null,institution:{name:"Universitas Internasional Batam",country:{name:"Indonesia"}}},{id:"314576",title:"Dr.",name:"Ibai",middleName:null,surname:"Laña",slug:"ibai-lana",fullName:"Ibai Laña",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314576/images/system/314576.jpg",biography:"Dr. Ibai Laña works at TECNALIA as a data analyst. He received his Ph.D. in Artificial Intelligence from the University of the Basque Country (UPV/EHU), Spain, in 2018. He is currently a senior researcher at TECNALIA. His research interests fall within the intersection of intelligent transportation systems, machine learning, traffic data analysis, and data science. He has dealt with urban traffic forecasting problems, applying machine learning models and evolutionary algorithms. He has experience in origin-destination matrix estimation or point of interest and trajectory detection. Working with large volumes of data has given him a good command of big data processing tools and NoSQL databases. He has also been a visiting scholar at the Knowledge Engineering and Discovery Research Institute, Auckland University of Technology.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"314575",title:"Dr.",name:"Jesus",middleName:null,surname:"L. Lobo",slug:"jesus-l.-lobo",fullName:"Jesus L. Lobo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314575/images/system/314575.png",biography:"Dr. Jesús López is currently based in Bilbao (Spain) working at TECNALIA as Artificial Intelligence Research Scientist. In most cases, a project idea or a new research line needs to be investigated to see if it is good enough to take into production or to focus on it. That is exactly what he does, diving into Machine Learning algorithms and technologies to help TECNALIA to decide whether something is great in theory or will actually impact on the product or processes of its projects. So, he is expert at framing experiments, developing hypotheses, and proving whether they’re true or not, in order to investigate fundamental problems with a longer time horizon. He is also able to design and develop PoCs and system prototypes in simulation. He has participated in several national and internacional R&D projects.\n\nAs another relevant part of his everyday research work, he usually publishes his findings in reputed scientific refereed journals and international conferences, occasionally acting as reviewer and Programme Commitee member. Concretely, since 2018 he has published 9 JCR (8 Q1) journal papers, 9 conference papers (e.g. ECML PKDD 2021), and he has co-edited a book. He is also active in popular science writing data science stories for reputed blogs (KDNuggets, TowardsDataScience, Naukas). Besides, he has recently embarked on mentoring programmes as mentor, and has also worked as data science trainer.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"103779",title:"Prof.",name:"Yalcin",middleName:null,surname:"Isler",slug:"yalcin-isler",fullName:"Yalcin Isler",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRyQ8QAK/Profile_Picture_1628834958734",biography:"Yalcin Isler (1971 - Burdur / Turkey) received the B.Sc. degree in the Department of Electrical and Electronics Engineering from Anadolu University, Eskisehir, Turkey, in 1993, the M.Sc. degree from the Department of Electronics and Communication Engineering, Suleyman Demirel University, Isparta, Turkey, in 1996, the Ph.D. degree from the Department of Electrical and Electronics Engineering, Dokuz Eylul University, Izmir, Turkey, in 2009, and the Competence of Associate Professorship from the Turkish Interuniversity Council in 2019.\n\nHe was Lecturer at Burdur Vocational School in Suleyman Demirel University (1993-2000, Burdur / Turkey), Software Engineer (2000-2002, Izmir / Turkey), Research Assistant in Bulent Ecevit University (2002-2003, Zonguldak / Turkey), Research Assistant in Dokuz Eylul University (2003-2010, Izmir / Turkey), Assistant Professor at the Department of Electrical and Electronics Engineering in Bulent Ecevit University (2010-2012, Zonguldak / Turkey), Assistant Professor at the Department of Biomedical Engineering in Izmir Katip Celebi University (2012-2019, Izmir / Turkey). He is an Associate Professor at the Department of Biomedical Engineering at Izmir Katip Celebi University, Izmir / Turkey, since 2019. In addition to academics, he has also founded Islerya Medical and Information Technologies Company, Izmir / Turkey, since 2017.\n\nHis main research interests cover biomedical signal processing, pattern recognition, medical device design, programming, and embedded systems. He has many scientific papers and participated in several projects in these study fields. He was an IEEE Student Member (2009-2011) and IEEE Member (2011-2014) and has been IEEE Senior Member since 2014.",institutionString:null,institution:{name:"Izmir Kâtip Çelebi University",country:{name:"Turkey"}}},{id:"339677",title:"Dr.",name:"Mrinmoy",middleName:null,surname:"Roy",slug:"mrinmoy-roy",fullName:"Mrinmoy Roy",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/339677/images/16768_n.jpg",biography:"An accomplished Sales & Marketing professional with 12 years of cross-functional experience in well-known organisations such as CIPLA, LUPIN, GLENMARK, ASTRAZENECA across different segment of Sales & Marketing, International Business, Institutional Business, Product Management, Strategic Marketing of HIV, Oncology, Derma, Respiratory, Anti-Diabetic, Nutraceutical & Stomatological Product Portfolio and Generic as well as Chronic Critical Care Portfolio. A First Class MBA in International Business & Strategic Marketing, B.Pharm, D.Pharm, Google Certified Digital Marketing Professional. Qualified PhD Candidate in Operations and Management with special focus on Artificial Intelligence and Machine Learning adoption, analysis and use in Healthcare, Hospital & Pharma Domain. Seasoned with diverse therapy area of Pharmaceutical Sales & Marketing ranging from generating revenue through generating prescriptions, launching new products, and making them big brands with continuous strategy execution at the Physician and Patients level. Moved from Sales to Marketing and Business Development for 3.5 years in South East Asian Market operating from Manila, Philippines. Came back to India and handled and developed Brands such as Gluconorm, Lupisulin, Supracal, Absolut Woman, Hemozink, Fabiflu (For COVID 19), and many more. In my previous assignment I used to develop and execute strategies on Sales & Marketing, Commercialization & Business Development for Institution and Corporate Hospital Business portfolio of Oncology Therapy Area for AstraZeneca Pharma India Ltd. Being a Research Scholar and Student of ‘Operations Research & Management: Artificial Intelligence’ I published several pioneer research papers and book chapters on the same in Internationally reputed journals and Books indexed in Scopus, Springer and Ei Compendex, Google Scholar etc. Currently, I am launching PGDM Pharmaceutical Management Program in IIHMR Bangalore and spearheading the course curriculum and structure of the same. I am interested in Collaboration for Healthcare Innovation, Pharma AI Innovation, Future trend in Marketing and Management with incubation on Healthcare, Healthcare IT startups, AI-ML Modelling and Healthcare Algorithm based training module development. I am also an affiliated member of the Institute of Management Consultant of India, looking forward to Healthcare, Healthcare IT and Innovation, Pharma and Hospital Management Consulting works.",institutionString:null,institution:{name:"Lovely Professional University",country:{name:"India"}}},{id:"1063",title:"Prof.",name:"Constantin",middleName:null,surname:"Volosencu",slug:"constantin-volosencu",fullName:"Constantin Volosencu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/1063/images/system/1063.png",biography:"Prof. Dr. Constantin Voloşencu graduated as an engineer from\nPolitehnica University of Timișoara, Romania, where he also\nobtained a doctorate degree. He is currently a full professor in\nthe Department of Automation and Applied Informatics at the\nsame university. Dr. Voloşencu is the author of ten books, seven\nbook chapters, and more than 160 papers published in journals\nand conference proceedings. He has also edited twelve books and\nhas twenty-seven patents to his name. He is a manager of research grants, editor in\nchief and member of international journal editorial boards, a former plenary speaker, a member of scientific committees, and chair at international conferences. His\nresearch is in the fields of control systems, control of electric drives, fuzzy control\nsystems, neural network applications, fault detection and diagnosis, sensor network\napplications, monitoring of distributed parameter systems, and power ultrasound\napplications. He has developed automation equipment for machine tools, spooling\nmachines, high-power ultrasound processes, and more.",institutionString:'"Politechnica" University Timişoara',institution:null},{id:"221364",title:"Dr.",name:"Eneko",middleName:null,surname:"Osaba",slug:"eneko-osaba",fullName:"Eneko Osaba",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/221364/images/system/221364.jpg",biography:"Dr. Eneko Osaba works at TECNALIA as a senior researcher. He obtained his Ph.D. in Artificial Intelligence in 2015. He has participated in more than twenty-five local and European research projects, and in the publication of more than 130 papers. He has performed several stays at universities in the United Kingdom, Italy, and Malta. Dr. Osaba has served as a program committee member in more than forty international conferences and participated in organizing activities in more than ten international conferences. He is a member of the editorial board of the International Journal of Artificial Intelligence, Data in Brief, and Journal of Advanced Transportation. He is also a guest editor for the Journal of Computational Science, Neurocomputing, Swarm, and Evolutionary Computation and IEEE ITS Magazine.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"275829",title:"Dr.",name:"Esther",middleName:null,surname:"Villar-Rodriguez",slug:"esther-villar-rodriguez",fullName:"Esther Villar-Rodriguez",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/275829/images/system/275829.jpg",biography:"Dr. Esther Villar obtained a Ph.D. in Information and Communication Technologies from the University of Alcalá, Spain, in 2015. She obtained a degree in Computer Science from the University of Deusto, Spain, in 2010, and an MSc in Computer Languages and Systems from the National University of Distance Education, Spain, in 2012. Her areas of interest and knowledge include natural language processing (NLP), detection of impersonation in social networks, semantic web, and machine learning. Dr. Esther Villar made several contributions at conferences and publishing in various journals in those fields. Currently, she is working within the OPTIMA (Optimization Modeling & Analytics) business of TECNALIA’s ICT Division as a data scientist in projects related to the prediction and optimization of management and industrial processes (resource planning, energy efficiency, etc).",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"49813",title:"Dr.",name:"Javier",middleName:null,surname:"Del Ser",slug:"javier-del-ser",fullName:"Javier Del Ser",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/49813/images/system/49813.png",biography:"Prof. Dr. Javier Del Ser received his first PhD in Telecommunication Engineering (Cum Laude) from the University of Navarra, Spain, in 2006, and a second PhD in Computational Intelligence (Summa Cum Laude) from the University of Alcala, Spain, in 2013. He is currently a principal researcher in data analytics and optimisation at TECNALIA (Spain), a visiting fellow at the Basque Center for Applied Mathematics (BCAM) and a part-time lecturer at the University of the Basque Country (UPV/EHU). His research interests gravitate on the use of descriptive, prescriptive and predictive algorithms for data mining and optimization in a diverse range of application fields such as Energy, Transport, Telecommunications, Health and Industry, among others. In these fields he has published more than 240 articles, co-supervised 8 Ph.D. theses, edited 6 books, coauthored 7 patents and participated/led more than 40 research projects. He is a Senior Member of the IEEE, and a recipient of the Biscay Talent prize for his academic career.",institutionString:"Tecnalia Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"278948",title:"Dr.",name:"Carlos Pedro",middleName:null,surname:"Gonçalves",slug:"carlos-pedro-goncalves",fullName:"Carlos Pedro Gonçalves",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRcmyQAC/Profile_Picture_1564224512145",biography:'Carlos Pedro Gonçalves (PhD) is an Associate Professor at Lusophone University of Humanities and Technologies and a researcher on Complexity Sciences, Quantum Technologies, Artificial Intelligence, Strategic Studies, Studies in Intelligence and Security, FinTech and Financial Risk Modeling. He is also a progammer with programming experience in:\n\nA) Quantum Computing using Qiskit Python module and IBM Quantum Experience Platform, with software developed on the simulation of Quantum Artificial Neural Networks and Quantum Cybersecurity;\n\nB) Artificial Intelligence and Machine learning programming in Python;\n\nC) Artificial Intelligence, Multiagent Systems Modeling and System Dynamics Modeling in Netlogo, with models developed in the areas of Chaos Theory, Econophysics, Artificial Intelligence, Classical and Quantum Complex Systems Science, with the Econophysics models having been cited worldwide and incorporated in PhD programs by different Universities.\n\nReceived an Arctic Code Vault Contributor status by GitHub, due to having developed open source software preserved in the \\"Arctic Code Vault\\" for future generations (https://archiveprogram.github.com/arctic-vault/), with the Strategy Analyzer A.I. module for decision making support (based on his PhD thesis, used in his Classes on Decision Making and in Strategic Intelligence Consulting Activities) and QNeural Python Quantum Neural Network simulator also preserved in the \\"Arctic Code Vault\\", for access to these software modules see: https://github.com/cpgoncalves. He is also a peer reviewer with outsanding review status from Elsevier journals, including Physica A, Neurocomputing and Engineering Applications of Artificial Intelligence. Science CV available at: https://www.cienciavitae.pt//pt/8E1C-A8B3-78C5 and ORCID: https://orcid.org/0000-0002-0298-3974',institutionString:"University of Lisbon",institution:{name:"Universidade Lusófona",country:{name:"Portugal"}}},{id:"310576",title:"Prof.",name:"Erick Giovani",middleName:null,surname:"Sperandio Nascimento",slug:"erick-giovani-sperandio-nascimento",fullName:"Erick Giovani Sperandio Nascimento",position:null,profilePictureURL:"https://intech-files.s3.amazonaws.com/0033Y00002pDKxDQAW/ProfilePicture%202022-06-20%2019%3A57%3A24.788",biography:"Prof. Erick Sperandio is the Lead Researcher and professor of Artificial Intelligence (AI) at SENAI CIMATEC, Bahia, Brazil, also working with Computational Modeling (CM) and HPC. He holds a PhD in Environmental Engineering in the area of Atmospheric Computational Modeling, a Master in Informatics in the field of Computational Intelligence and Graduated in Computer Science from UFES. He currently coordinates, leads and participates in R&D projects in the areas of AI, computational modeling and supercomputing applied to different areas such as Oil and Gas, Health, Advanced Manufacturing, Renewable Energies and Atmospheric Sciences, advising undergraduate, master's and doctoral students. He is the Lead Researcher at SENAI CIMATEC's Reference Center on Artificial Intelligence. In addition, he is a Certified Instructor and University Ambassador of the NVIDIA Deep Learning Institute (DLI) in the areas of Deep Learning, Computer Vision, Natural Language Processing and Recommender Systems, and Principal Investigator of the NVIDIA/CIMATEC AI Joint Lab, the first in Latin America within the NVIDIA AI Technology Center (NVAITC) worldwide program. He also works as a researcher at the Supercomputing Center for Industrial Innovation (CS2i) and at the SENAI Institute of Innovation for Automation (ISI Automação), both from SENAI CIMATEC. He is a member and vice-coordinator of the Basic Board of Scientific-Technological Advice and Evaluation, in the area of Innovation, of the Foundation for Research Support of the State of Bahia (FAPESB). He serves as Technology Transfer Coordinator and one of the Principal Investigators at the National Applied Research Center in Artificial Intelligence (CPA-IA) of SENAI CIMATEC, focusing on Industry, being one of the six CPA-IA in Brazil approved by MCTI / FAPESP / CGI.br. He also participates as one of the representatives of Brazil in the BRICS Innovation Collaboration Working Group on HPC, ICT and AI. He is the coordinator of the Work Group of the Axis 5 - Workforce and Training - of the Brazilian Strategy for Artificial Intelligence (EBIA), and member of the MCTI/EMBRAPII AI Innovation Network Training Committee. He is the coordinator, by SENAI CIMATEC, of the Artificial Intelligence Reference Network of the State of Bahia (REDE BAH.IA). He leads the working group of experts representing Brazil in the Global Partnership on Artificial Intelligence (GPAI), on the theme \"AI and the Pandemic Response\".",institutionString:null,institution:null},{id:"241400",title:"Prof.",name:"Mohammed",middleName:null,surname:"Bsiss",slug:"mohammed-bsiss",fullName:"Mohammed Bsiss",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241400/images/8062_n.jpg",biography:null,institutionString:null,institution:null},{id:"276128",title:"Dr.",name:"Hira",middleName:null,surname:"Fatima",slug:"hira-fatima",fullName:"Hira Fatima",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/276128/images/14420_n.jpg",biography:"Dr. Hira Fatima\nAssistant Professor\nDepartment of Mathematics\nInstitute of Applied Science\nMangalayatan University, Aligarh\nMobile: no : 8532041179\nhirafatima2014@gmal.com\n\nDr. Hira Fatima has received his Ph.D. degree in pure Mathematics from Aligarh Muslim University, Aligarh India. Currently working as an Assistant Professor in the Department of Mathematics, Institute of Applied Science, Mangalayatan University, Aligarh. She taught so many courses of Mathematics of UG and PG level. Her research Area of Expertise is Functional Analysis & Sequence Spaces. She has been working on Ideal Convergence of double sequence. She has published 17 research papers in National and International Journals including Cogent Mathematics, Filomat, Journal of Intelligent and Fuzzy Systems, Advances in Difference Equations, Journal of Mathematical Analysis, Journal of Mathematical & Computer Science etc. She has also reviewed few research papers for the and international journals. She is a member of Indian Mathematical Society.",institutionString:null,institution:null},{id:"417317",title:"Mrs.",name:"Chiedza",middleName:null,surname:"Elvina Mashiri",slug:"chiedza-elvina-mashiri",fullName:"Chiedza Elvina Mashiri",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Midlands State University",country:{name:"Zimbabwe"}}},{id:"352140",title:"Dr.",name:"Edina",middleName:null,surname:"Chandiwana",slug:"edina-chandiwana",fullName:"Edina Chandiwana",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Midlands State University",country:{name:"Zimbabwe"}}},{id:"342259",title:"B.Sc.",name:"Leonard",middleName:null,surname:"Mushunje",slug:"leonard-mushunje",fullName:"Leonard Mushunje",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Midlands State University",country:{name:"Zimbabwe"}}},{id:"347042",title:"Mr.",name:"Maxwell",middleName:null,surname:"Mashasha",slug:"maxwell-mashasha",fullName:"Maxwell Mashasha",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Midlands State University",country:{name:"Zimbabwe"}}},{id:"2941",title:"Dr.",name:"Alberto J.",middleName:"Jorge",surname:"Rosales-Silva",slug:"alberto-j.-rosales-silva",fullName:"Alberto J. Rosales-Silva",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"437913",title:"Dr.",name:"Guillermo",middleName:null,surname:"Urriolagoitia-Sosa",slug:"guillermo-urriolagoitia-sosa",fullName:"Guillermo Urriolagoitia-Sosa",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"435126",title:"Prof.",name:"Joaquim",middleName:null,surname:"José de Castro Ferreira",slug:"joaquim-jose-de-castro-ferreira",fullName:"Joaquim José de Castro Ferreira",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Aveiro",country:{name:"Portugal"}}},{id:"437899",title:"MSc.",name:"Miguel Angel",middleName:null,surname:"Ángel Castillo-Martínez",slug:"miguel-angel-angel-castillo-martinez",fullName:"Miguel Angel Ángel Castillo-Martínez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"289955",title:"Dr.",name:"Raja",middleName:null,surname:"Kishor Duggirala",slug:"raja-kishor-duggirala",fullName:"Raja Kishor Duggirala",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Jawaharlal Nehru Technological University, Hyderabad",country:{name:"India"}}}]}},subseries:{item:{id:"27",type:"subseries",title:"Multi-Agent Systems",keywords:"Collaborative Intelligence, Learning, Distributed Control System, Swarm Robotics, Decision Science, Software Engineering",scope:"Multi-agent systems are recognised as a state of the art field in Artificial Intelligence studies, which is popular due to the usefulness in facilitation capabilities to handle real-world problem-solving in a distributed fashion. The area covers many techniques that offer solutions to emerging problems in robotics and enterprise-level software systems. Collaborative intelligence is highly and effectively achieved with multi-agent systems. Areas of application include swarms of robots, flocks of UAVs, collaborative software management. Given the level of technological enhancements, the popularity of machine learning in use has opened a new chapter in multi-agent studies alongside the practical challenges and long-lasting collaboration issues in the field. It has increased the urgency and the need for further studies in this field. We welcome chapters presenting research on the many applications of multi-agent studies including, but not limited to, the following key areas: machine learning for multi-agent systems; modeling swarms robots and flocks of UAVs with multi-agent systems; decision science and multi-agent systems; software engineering for and with multi-agent systems; tools and technologies of multi-agent systems.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/27.jpg",hasOnlineFirst:!0,hasPublishedBooks:!1,annualVolume:11423,editor:{id:"148497",title:"Dr.",name:"Mehmet",middleName:"Emin",surname:"Aydin",slug:"mehmet-aydin",fullName:"Mehmet Aydin",profilePictureURL:"https://mts.intechopen.com/storage/users/148497/images/system/148497.jpg",biography:"Dr. Mehmet Emin Aydin is a Senior Lecturer with the Department of Computer Science and Creative Technology, the University of the West of England, Bristol, UK. His research interests include swarm intelligence, parallel and distributed metaheuristics, machine learning, intelligent agents and multi-agent systems, resource planning, scheduling and optimization, combinatorial optimization. Dr. Aydin is currently a Fellow of Higher Education Academy, UK, a member of EPSRC College, a senior member of IEEE and a senior member of ACM. In addition to being a member of advisory committees of many international conferences, he is an Editorial Board Member of various peer-reviewed international journals. 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