Astaxanthin in animal feed and aquatic feed
Astaxanthin (ATX) is currently considered the second most important carotenoid in terms of global market value, a red-orange fat-soluble diketocarotenoid. Astaxanthin is a valuable pigment known for its outstanding antioxidant activity and for its many biological activities. It is widely used in various industries including feed, food, pharmaceutical, nutritional and cosmetics. For many years, it has been used as a feed additive in aquaculture and poultry farming to increase the color of the meat of farm-raised aquatic animals and the eggs of poultry. With its significant impact on animal health and nutrition, the use of astaxanthin as a supplement in animal feed has also been extended to livestock. Synthetic astaxanthin is by far the main form of astaxanthin used in animal feed. However, with the recent interest in natural astaxanthin, the exploration and exploitation of microbial sources of ATX has received great attention. Among the microbial sources exploited is astaxanthin bacteria (the least developed and least used). Therefore, this review considers bacterial astaxanthin and its use as a supplement in animal feed.
INTRODUCE
Introduction Carotenoids are a common group of essential compounds in nature, found in both plants and animals. They function primarily as pigments in plants, algae, and other photosynthetic organisms. Additionally, carotenoids are important compounds in animals, with a multitude of biological functions and activities including antioxidant and provitamin A activity (1-3). Accordingly, they have received considerable attention across all industries for many years, especially in the feed and food industry as colorants for colored beverages, dairy products and meat (4).
When choosing a food item (e.g. meat) from among available options, color is an important subconscious factor. For example, the red color of some fish species is a distinctive feature that adds value to the resulting products giving them a higher market value (5). Therefore, carotenoids are included in animal feed to improve animal health and, most importantly, product quality. Today, carotenoids are produced commercially for animal feed and have a total market value exceeding $1.5 billion (3, 6). The main carotenoids used in animal feed include astaxanthin, β-carotene, lutein, zeaxanthin, canthaxanthin and capsanthin (6).
Notably the xanthophyll carotenoid, astaxanthin (3,3′-dihydroxylate and 4,4′-diketolate derivative of β-carotene), occupies a special place among them as the most powerful antioxidant. Astaxanthin is a lipid-soluble, orange-red diketocarotenoid found primarily in the marine environment. It consists of a polyene chain sandwiched between two terminal β-ionone rings, each bearing a characteristic 3-hydroxyl and 4-keto moiety (7).
This unique structure gives it optimal orientation within the membrane (8). Astaxanthin is mainly synthesized by microalgae, yeast, bacteria and some plants (9). Although not generally synthesized by animals, it is found in various marine animals (especially salmonids; and crustaceans such as shrimp, crabs, lobsters, and crayfish). and some birds (such as flamingos and quail) due to direct accumulation from algae as well as zooplankton or insects (which consume the main production source of astaxanthin) (10, 11).
In many of these aquatic animals, in addition to pigmentation, ATX has several essential biological roles including protection against oxidation of essential polyunsaturated fatty acids; protects against the effects of UV rays; immune response; communicate; reproductive behavior and improve fertility (11). Therefore, due to its ability to accumulate in various animal structures such as skin, muscles, eggs, etc. ATX is added to animal feed to pigment egg yolks, broiler skin, fish meat and crustaceans, to enhance its commercial value and general consumer acceptance. (6, 10). Astaxanthin is especially known for its use in aquaculture to pigment salmon meat, accounting for 15-20% of feed costs (1, 12). Globally, it is considered the second most important carotenoid by market value, with a global market worth more than $600 million in 2018 (13, 14).
To date, chemical synthesis (from petrochemicals) remains the main source of astaxanthin, especially in the animal feed industry, providing up to 95% of commercially available astaxanthin globally (10). This is largely because chemical synthesis is by far the cheapest production process for ATX. However, there is a growing consumer demand for natural foods and additives due to growing awareness of the harmful effects and potential harm of chemically synthesized additives as well as restrictions on their use. by law in some countries, has led to an increase in interest and investment in nature. source of (ATX) (5).
Furthermore, unlike synthetic (ATX), which is a mixture of three stereoisomers: (3S, 3ʹS), (3S, 3ʹR) and (3R, 3ʹR) in a 1:2:1 ratio, biogenic ATX More stereoselective synthesis leads to the production of enantiostatically pure astaxanthin, (3S, 3ʹS) or (3R, 3ʹR), which is desirable for human use and largely considered biologically active. excel in learning (9, 15, 16). Relatively few papers have reported the stereo profiles of bacterial ATX While the yeast X. dendrorhous is well known as the only natural producer of (3R, 3ʹR) ATX, microalgae and bacteria (at least (especially those species isolated to date) mainly produce (3S, 3ʹS).
Although stereoisomers are generally known to have different biological activities, there has been no in-depth study of the differences in biological activity between (3S, 3ʹS) and (3R, 3ʹR). Astaxanthin is widely known to be highly sensitive to heat, intense light, and oxidative conditions due to its highly unsaturated molecular structure (10). Therefore, exposure of astaxanthin to this range of conditions during extraction from natural sources with complex matrices is undesirable – because of the significant amount of pigment and therefore the desired biological effect. will be lost. For this reason and many other advantages, microbial sources are of particular interest as natural sources of astaxanthin.
The main commercial microbial sources of astaxanthin include algae, yeast, and bacteria. Although algae and yeast are the main organisms used for the industrial production of astaxanthin, these two have thicker cell walls and therefore homogenization and cell disruption are necessary to increase the pigment accessibility in the animal’s digestive tract (4). As reported by (17-19), less pigmentation was observed when whole yeast or algal cells were used.
However, disruption of cells inevitably causes astaxanthin to undergo thermal, photometric, and oxidative degradation. In this regard, bacterial astaxanthin stands out for being easily absorbed from whole cells – without the need for cell disruption (20). In addition, bacteria have certain unique advantages of yeast and algae including shorter life cycles, compatibility with seasons and climate, and greater ease of scaling (21). Despite these advantages, bacterial astaxanthin production still lags significantly behind algae (the dominant source of astaxanthin production today) and yeast in terms of astaxanthin content in biomass.
Therefore, not surprisingly, bacterial astaxanthin or its products are commercially scarce. In fact, at the time of compilation of this review, only JX Nippon ANCI was found to be producing bacterial ATX (Panaferd®) in commercial quantities. However, some bacteria have been identified as capable of producing astaxanthin, e.g., Paracoccus spp., Agrobacteria spp., Sphingomonas spp., etc. Additionally, to optimize bacterial astaxanthin production, non-native bacterial producers have also been engineered to produce astaxanthin, some of which include: Methylomonas sp. (22), Corynebacteria glutamicum (23), E. coli (24). Although there is a wealth of literature on astaxanthin, its production and applications, not much attention has been paid to bacterial ATX. Therefore, this chapter reviews bacterial production of astaxanthin and the use of bacterial astaxanthin as a feed supplement.
Indigenous Bacterial Producers of Astaxanthin
The rapidly growing demand for (ATX), especially from natural sources, has motivated scientists and researchers to search for and isolate new astaxanthin-producing bacteria. Therefore, several new astaxanthin-producing microorganisms, especially bacteria, have been isolated specifically from the marine environment (25).
To date, different types of bacteria, Gram-positive (e.g., Brevundimonas spp.) and Gram-negative (e.g., Paracoccus ssp.), have been identified as capable of producing astaxanthin (15). Among them is Paracoccus ssp. (including: P. carotenifaciens, P. marcusii, P. haeundaensis, P. bogoriensis and other strains) is one of the most promising bacterial groups for commercial astaxanthin production (26). Cyanobacteria are another type of bacteria with promising potential for commercial astaxanthin production. Several astaxanthin producers have been isolated from this group; Geitlerimena amphibium, Synechococcus spp., Phormidium spp., Osillatoria subbrevis and many others (15).
Equally prominent astaxanthin-producing bacteria belong to the genus Brevundimonas. Strains isolated from this genus to date include B. aurantiaca, B. vesicularis, B. bacteroides (15). In addition, several other non-marine bacteria including Sphingomonas astaxanificiens and Sphingomonas faeni have also been identified as capable of producing astaxanthin (27). The astaxanthin content in these bacteria varies widely, however, in most of them, the amount of astaxanthin is too small to be scaled up on an industrial scale (Table 1). However, studies on bacterial astaxanthin synthesis are of great significance for astaxanthin production because success in this area is expected to significantly reduce the cost of natural astaxanthin production.
It is worth noting that in addition to the common forms of (ATX), some bacterial isolates have also been shown to produce various derivatives of astaxanthin including hydroxylated and glycosylated derivatives (Figure 1). Currently, astaxanthin dirhamnoside from S. astaxanthinifaciens (28), astaxanthin glucoside from A. aurantiacum (29), astaxanthin dideoxyglycoside from Sphingomonas sp. (30), 2-Hydroxy-astaxanthin and 2,2′-Dihydroxy-astaxanthin from Brevundimonas sp. (25, 31, 32) have all been reported in the literature. Although these derivatives have not been evaluated in food and feed, they may contribute significantly to the overall effectiveness of using whole bacteria as a source of astaxanthin.
Probiotic manufacturers of Astaxanthin
Although to date, a number of bacteria have been identified as astaxanthin producers, commercial astaxanthin production from these bacteria has not yet been established – with the exception of P. carotinifaciens which is currently used for its production. Panaferd® by JX Nippon ANCI, Inc. (60) This is most likely due to the unsatisfactory amount of astaxanthin they produce. Therefore, in addition to searching for new bacteria with higher astaxanthin productivity, some non-astaxanthin-producing bacteria (including Escherichia coli, Corynebacter glutamicum, etc.) have been engineered to produce (ATX) commercially to meet meet growing demand. of natural astaxanthin (15).
Thanks to advances in biotechnology, identifying and cloning the genes involved in astaxanthin synthesis is one of the key techniques to transform these bacteria and other organisms into astaxanthin producers.
Additionally, mutagenesis has been applied to improve astaxanthin production by these modified organisms (15, 61). Gene clusters (Figure 2) involved in astaxanthin synthesis have so far been isolated and characterized from several organisms including the common natural astaxanthin sources H. pluvialis and X. dendrorhous; Paracoccus spp.; cyanobacteria; Brevundimonas spp.; C. zofingiensis as well as from the plant (A. aestival) (15, 61, 62).
E.coli is one of the bacteria widely genetically engineered to produce astaxanthin — perhaps because it can be grown at high densities using well-established fermentation techniques (15, 24, 61). Carotenogenic bacteria, such as C. glutamicum (which is not naturally an astaxanthin producer) have also been engineered to produce astaxanthin using genes from different organisms (Table 2) (23, 63, 64). Other unusual bacteria such as Rhodovulum sulfidophilum, Methylomonas spp. In addition, they are also used as hosts to produce astaxanthin through the astaxanthin biosynthetic pathway (65, 66).
While some of these transformations are just a proof of concept, others have been optimized for higher yields – with some achieving astaxanthin yields comparable to H. pluvialis and X. dendrorhous.
Figure 2. (ATX) synthesis gene clusters from different organisms (Black clusters are from algae; green clusters are from bacteria; blue clusters are from fungi (X. dendrorhous); red clusters are Note: the letter before “Crt” indicates the organism containing the gene. For example, SpcCrtZ and SprCrtZ indicate the CrtZ gene of S. paucimobileis and S. parapaucimobileis.
Bacterial astaxanthin as an animal feed supplement
Historically, livestock farming involved fresh food in the wild – the traditional extensive livestock system. However, gradually this has been almost completely replaced by intensive and semi-intensive systems due to increasing commercial demand for animal products (5, 6). Optimal animal nutrition is paramount in the following systems and therefore the industry has found ways to formulate feeds to provide animals with the necessary nutrients. One such way is supplementing feed with various additives.
For many years, carotenoids have been included in animal diets to support growth as well as product quality (6). They have been used successfully in poultry and aquaculture (the two main markets for carotenoid feed additives) to modify the color of egg yolks, fish meat and crustaceans.
They have also been used in the skin pigmentation of ornamental fish (70). One of the main carotenoids used in the animal feed industry is (ATX). In addition to pigmentation, astaxanthin also reduces lipid peroxidation, improving fertility and reproduction, overall egg quality, and survival of young animals (5, 20).
Following the general trend of carotenoids in the feed industry, more than 90% of astaxanthin used as feed additives is chemically synthesized. Given that animal feed is at the top of the food chain, growing public concern, especially about synthetic chemical additives, has led to strict legal regulation of carotenoids used in feed. feed livestock (71). Although over the years suitable astaxanthin (algal and bacterial yeast) producers have been identified, they currently only serve a niche market (especially the pharmaceutical and nutritional industries) due to Production costs are higher than chemical synthesis (70).
However, natural astaxanthin is being exploited as an animal feed additive, with its use increasing. To avoid the complicated extraction process as well as loss of potency, the use of whole cells (biomass) containing astaxanthin has attracted attention as a viable alternative to astaxanthin supplementation in dietary regimens. animal food. In this regard, bacterial cells have an advantage because they have thinner cell walls. However, compared to algae and yeast, bacterial astaxanthin is used only sparingly in animal feed across all sectors including poultry and aquaculture.
Astaxanthin bacteria in poultry
Chickens have become one of the most domesticated animals, raised for meat and eggs (4). Egg yolks and well-pigmented poultry meat have long been associated with good health. In many countries, the bright yellow or orange-yellow color of egg yolks as well as the meat and skin of broiler chickens is considered by consumers to be a sign of freshness and healthiness and has therefore become a quality indicator ( 6).
Despite this, consumer preferences and acceptability regarding color vary depending on geographical location (e.g., while consumers in Germany, Spain and Belgium prefer egg yolks was more orange, consumers in Ireland, Sweden and Southern England wanted it to be lighter in color) (72). The composition and concentration of carotenoids in feed are among the factors that influence the pigmentation of poultry and its products (6).
In general, pigmentation in poultry is thought to be due to the accumulation of specific xanthophylls (71). Due to their low absorption capacity (due to low polarity) as well as their ability to convert to vitamin A, β-carotene and other carotenoids (e.g., carotenes) contribute little to poultry pigmentation (72, 73 ). Consequently, xanthophylls have gained economic interest in coloring the skin of broiler chickens and especially egg yolks with the main xanthophylls used in modern poultry being lutein, zeaxanthin and canthaxanthin (71).
Due to the transfer of carotenoids from poultry to humans in meat and eggs, in addition to the fact that astaxanthin is a super carotenoid, the use of ATX (especially from natural sources) in poultry production is gradually being appreciated. . Astaxanthin is reported to produce pinkish egg yolks when used alone, which is undesirable for most consumers in the EU (71).
However, when used in combination with other xanthophylls, it is said to increase pigmentation 30 times more than lutein (72). Following the growing demand for more natural products, several natural sources of astaxanthin including bacteria have been evaluated in poultry production. Feeding laying hens P. marcussi biomass containing astaxanthin, in freeze-dried form or live cells, resulted in significantly increased egg production. Additionally, significant increases in egg quality parameters including overall egg weight, yolk weight and yolk color were observed.
However, no significant differences were observed in the body weight of hens (4). In addition, the feed formula with Paracoccus bacterial cells (with 2% astaxanthin content according to DCW) significantly increased the red color of chicken eggs compared to the feed formula with chili powder as a color source (74). In addition to egg yolk pigmentation, supplementation of the basal diet with 0.15% astaxanthin-rich dry powder of P. carotinifaciens significantly increased the red color of broiler meat, especially breast and leg muscles (75 ). Unlike P. rhodozyma biomass (76) In addition, P. carotinifaciens biomass also increases the yellowness of broiler meat. It also leads to an increase in carotenoid content in meat by accumulation of carotenoids derived from P. carotinifaciens (e.g., ATX, adonixanthin and canthaxanthin) as well as increased deposition of other carotenoids present in the diet. (e.g. zeaxanthin and lutein).
Malondialdehyde, MDA, (a biomarker of oxidative stress and lipid peroxidation) was significantly reduced in diets supplemented with biomass rich in astaxanthin (75). Chickens are known to be susceptible to heat stress (a condition that occurs at ambient temperatures above the thermoneutral zone). Heat stress is a major concern in poultry production because it adversely affects growth performance, body weight, and carcass characteristics such as pigmentation (77).
Supplementing P. carotinifaciens powder significantly improved heat-induced hypopigmentation. However, supplementation did not improve body weight or food intake that was reduced by heat stress (75). Toyomizu et al. (78) in evaluating the effect of dietary spirulina on broiler meat pigmentation observed that the red color value of meat increased with spirulina supplementation.
At that time, the authors did not draw firm conclusions about the cause of the increased red color of broiler meat in the supplemented group. Currently, it has been reported that spirulina produces astaxanthin as part of the carotenoid (51, 52). Therefore, the increase in red color value of broiler meat observed by (78) may be due to (ATX). However, the use of bacterial astaxanthin in commercial poultry production is still limited.
Bacterial astaxanthin in aquaculture
Astaxanthin is one of the most abundant marine carotenoids. Most marine and freshwater fish as well as other aquatic organisms, such as crustaceans, have their bright pink-red color thanks to astaxanthin (71).
In nature, carotenoids (e.g. astaxanthin) in crustaceans come mainly from algae while in fish, from plankton or other fish that have crustaceans in their digestive tract (6). The color of fish flesh or the exoskeleton of crustaceans is an important quality parameter that influences customer choice and ultimately the market value of fish and crustaceans (71). These aquatic animals are generally unable to synthesize ATX de novo (10).
Therefore, to meet consumer needs for flesh pigmentation and the exoskeleton of fish or crustaceans, diets in intensive aquaculture are supplemented with astaxanthin and other oxycarotenoids (6). The continuous advancement of the aquaculture industry has created a great demand for astaxanthin as a colorant (10). This is the oldest and probably the largest (by volume) consumer of astaxanthin (especially synthetic ATX).
Although primarily used as a pigment in aquaculture, astaxanthin provides additional benefits to farmed marine animals thanks to its broad biological activities. In salmon, it has been reported to improve immunity and resistance to bacterial and fungal diseases while in crustaceans, it significantly increases survival rates (6). Currently, several natural sources of astaxanthin are used in industrial aquaculture, among the mined sources include algae, yeast, shrimp waste and (least used) bacterial cells.
Astaxanthin from algae and yeast has been well developed industrially, however, their use as a substitute for synthetic ATX has proven unsuccessful partly due to its relatively expensive nature. Therefore, bacterial astaxanthin is being considered as an alternative source but the development of this source of astaxanthin is still at a major research stage with varying success. Supplementing P. marcusii (live cells) significantly increased the growth rate of Apostichopus japonicus (sea cucumber).
It also improved the immune response of sea cucumbers, which was noted to have higher phagocytic activity of coelomocytes as well as increased superoxide dismutase and lysozyme activity of coelomocytes (79). A previous study also reported that live cell supplementation of P. marcusii significantly improved the growth performance and immune response of young A. japonicus (80). Additionally, dry powder of P. carotinifaciens (Panaferd-AX®) dose-dependently increased the amount of yellow xanthophylls in the muscle, head, and carapace of Penaeus japonicus when added to their diet (81).
However, the addition of Paracoccus sp. biomass in the diet of Red Sea Bream did not increase growth rate but significantly increased ATX concentration as well as total carotenoids in the skin (82). Similarly, in a previous study, no significant differences were observed in the specific growth rates of Red Sea Bream fed with synthetic ATX, P. rhodozyma biomass or Paracoccus sp. biomass. The group was fed diet containing Paracoccus sp. however, the biomass recorded the highest levels of astaxanthin and total carotenoids in the skin, a difference that was especially pronounced when compared with the group fed a diet supplemented with synthetic ATX. Accordingly, the skin of the group fed with natural ATX (from P. rhodozyma and Paracoccus sp.) was reported to be redder, although the authors did not indicate which of the two provided skin pigmentation. better for red sea bream – probably Paracoccus sp. group was supplemented because astaxanthin and the highest total skin carotenoid content were observed in this group (83).
In aquarium fish, P. carotinifaciens (Panaferd-AX®) was observed to increase the red coloration of the anal fin and anterior dorsal region of Puntius titteya. The authors, quite interested in the effects of astaxanthin on fish behavior, reported that the addition of Panaferd-AX® reduced the strong interaction of male P. titteya with the mirror image. Additionally, it was reported that under UV-blocked light spectrum conditions, males spent more time with females fed a diet supplemented with P. carotinifaciens (containing 20ppm astaxanthin). Supplementation with P. carotinifaciens (40 ppm ATX) also increased the time males spent with females under full spectrum light, but not as much as those raised with synthetic astaxanthin (84).
According to the report of the EU panel established to evaluate the safety and effectiveness of Panaferd-AX® (astaxanthin-rich powder of P. carotinifaciens), Coho salmon fed with Panaferd-AX® fortified diets also noted slightly higher weight. Meat astaxanthin content was higher than that of the synthetic ATX group in a preliminary study. However, in a large-scale trial, astaxanthin deposition in the skin was significantly higher in the group fed with synthetic ATX. Additionally, when evaluating the color of the meat (via color fans), there was no significant difference between the two sources of astaxanthin. At the same astaxanthin concentration level, no significant difference in red color value was observed. Similar results were reported when research was performed on salmon, although astaxanthin accumulation in the skin and meat was much lower (60).
In a separate study with Atlantic salmon, no significant differences were observed in the specific growth rates of fish fed with synthetic ATX (Carophyll® pink) and bacterial astaxanthin (Panaferd- AX®). Although the total carotenoid content in the feed with bacterial astaxanthin was almost twice that of the synthetic ATX, no such large differences were observed in the total carotenoid content of the fillets, with the group with bacterial astaxanthin being the highest. In terms of astaxanthin deposition in meat, synthetic ATX resulted in significantly higher astaxanthin content in meat, although ATX content in each food was similar in both groups. Red values were similar in both groups (85).
Other small bacterial sources of astaxanthin, such as the cyanobacterium Spirulina, have also been exploited in aquaculture to influence: growth, immunity, proximate composition and pigmentation of salmon (86 -89), growth performance and total carotenoid content of Barilius bentelisis (90) as well as pigmentation of ornamental fish (91). Although each of these studies did not focus on astaxanthin because it is not one of the main carotenoids in Spirulina. However, it has been shown that astaxanthin significantly increases the deposition of other carotenoids in the meat and skin of aquatic animals (75), hence the role of astaxanthin in the overall effects of Spirulina. in these experiments (although not evaluated by the authors) overlooked.
Astaxanthin bacteria in livestock
Agriculture Carotenoids are a very important group of compounds, not only in plants (where they are mainly synthesized) but also in animals. Extensive research has demonstrated a variety of important biological roles for carotenoids in animals, from serving as integral members of cell membranes to improving the general health of animals (1-3). However, it seems that not much attention is paid to carotenoids in industrial livestock (e.g. Cows, sheep, goats, pigs, etc.). To date, there have been only a few studies evaluating dietary supplementation of ATX in livestock such as cattle, goats, sheep and the like. In buffalo, ATX supplementation was reported to increase milk yield while ameliorating the adverse effects of heat stress (92). Likewise, it has been shown to prevent heat stress in cattle. As in poultry, heat stress is a major factor affecting productivity in livestock production – adversely affecting growth performance as well as milk and meat production (93). Additionally, ATX supplementation in suckling lambs has been reported to reduce hydroxytoluene accumulation in finished meat and slightly increase the red color of meat and fat (94). However, at the time of compiling this article, no evidence of the use of astaxanthin bacteria in livestock production was found.
Conclusion
Consumer demand for well-pigmented and fortified animal products is driving the growing use of carotenoids such as Astaxanthin in the feed industry. ATX is an important xanthophyll that has attracted significant interest in several industries, especially as a colorant in the feed and food industries. It is responsible for the bright and attractive pink-red color of most aquatic animals. It is an economically and industrially essential component of poultry feed and aquaculture for pigmenting egg yolks, meat or skin or poultry and fish. In addition, it provides many benefits to livestock including improving growth performance, survival, reproductive physiology, stress tolerance, disease resistance and general immunity. . Over several decades there has been a dramatic shift in the food industry driven by consumer preferences for more natural and organic products. In response to the growing trend, various natural sources have been evaluated and explored as alternatives to synthetic astaxanthin. Among them, bacterial ATX is least developed and used. Currently strategies such as genetic and metabolic engineering along with improved fermentation technologies are being used to increase bacterial ATX production. Bacterial astaxanthin, in terms of pigmentation and other benefits, stands convincingly against synthetic ATX, the type most used today. However, for bacterial ATX to compete economically with synthetic astaxanthin in the global market, its current high price as well as other microbial sources must be reduced. Therefore, it is necessary to continue research on ATX bacteria: identify or develop super-producing bacteria as well as develop sustainable and economical ATX production processes.
Reference source: Astaxanthin from bacteria as a feed supplement for animals
Osman N. Kanwugua, Ambati Ranga Raob, Gokare A. Ravishankarc, Tatiana V. Glukharevaa,
Elena G. Kovalevaa*,
1Ural Federal University named after the first President of Russia B.N. Yeltsin, Mira street 19,
620002 Ekaterinburg, Russia
bCentre of Excellence, Department of Biotechnology, Vignan’s Foundation for Science,
Technology and Research (Deemed to be University), Vadlamudi, Guntur, Andhra Pradesh, Indi
cC.D. Sagar Centre for Life Sciences, Dayananda Sagar College of Engineering, Dayananda
Sagar Institutions, Bangalore, Karnataka, India