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Integration of protein extraction with a stream of byproducts from marine macroalgae: A model forms the basis for marine bioeconomy
Accepted Manuscript Integration of protein extraction with a stream of byproducts from marine macroalgae: a model forms the basis for marine bioeconomy Tejal K. Gajaria, Poornima Suthar, Ravi S. Baghel, Nikunj B.Balar, Preeti Sharnagat, Vaibhav A. Mantri, C.R.K. Reddy PII: DOI: Reference:
S0960-8524(17)31053-2 http://dx.doi.org/10.1016/j.biortech.2017.06.149 BITE 18386
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
29 May 2017 24 June 2017 26 June 2017
Please cite this article as: Gajaria, T.K., Suthar, P., Baghel, R.S., B.Balar, N., Sharnagat, P., Mantri, V.A., Reddy, C.R.K., Integration of protein extraction with a stream of byproducts from marine macroalgae: a model forms the basis for marine bioeconomy, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.06.149
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Integration of protein extraction with a stream of byproducts from marine macroalgae: a model forms the basis for marine bioeconomy Tejal K. Gajaria1,2, Poornima Suthar1, Ravi S. Baghel1,2, Nikunj B.Balar1,2, Preeti Sharnagat1, Vaibhav A. Mantri1,2 and C.R.K. Reddy1,2 *
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Division of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine
Chemicals Research Institute, Bhavnagar-364002, India 2
Academy of Scientific and Innovative Research (AcSIR), New Delhi, India
Abstract The present study describes an advanced biorefinery model for marine macroalgae that assumes significant importance in the context of marine bio-economy. The method investigated in this study integrates the extraction of crude proteins with recovery of minerals rich sap, lipids, ulvan and cellulose from fresh biomass of Ulva lactuca. The protein content extracted was 11±2.12 % on dry weight basis with recovery efficiency of 68.75±4.01%. The amino acid composition of crude protein fraction showed isoleucine as the most abundant amino acid with 16.51±0.03% followed by histidine, arginine, tyrosine, serine, aspartic acid, threonine, phenyl alanine, leucine, alanine, lysine, glycine and glutamic acid (0.22±0.24%). The digestibility of protein was as high as 85.86±5.92% indicating its suitability for use in food supplements. The protein production with co-recovery of other products would not only result in effective utilisation marine macroalgal resources but also forms the basis for marine bioeconomy.
Keywords: Algal salt, Integrated biorefinery, Nutritional supplement, Sustainable process, Ulvan *Author for correspondence Tel: +91 278 257 0885 Fax: + 91 278 256 6970 / 256 7562 Email: [email protected]
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1. Introduction Marine macroalgae, commonly known as seaweeds, comprise of assemblage of diverse autotrophic lower plants that are not only integral part of oceanic ecosystem but also play an important role in CO2 sequestration besides rendering invaluable ecological services. Seaweeds have historically been used as part of human diets in some Fareast countries (Holdt and Kraan, 2011) while in other parts of the world they are used as a source of raw material for extraction of industrially import hydrocolloids such as agar, carrageenan and alginates (Bixler and Porse, 2011). In recent times, seaweeds have also been gaining considerable importance globally as non-lignocellulosic feedstock for production of bioenergy (Jang et al., 2012). The macroalgal biorefinery is one of the rapidly emerging frontier research areas of bio-processing for a sustainable production of variety of biomolecules. It possesses immense potential to provide possible alternatives to both global environmental concerns and a noncompetitive green source for production of nutrient supplements as well as other commodity materials. Bio-refinery utilises biomass components that are synthesised as a function of complex photosynthesis and therefore they have to be effectively processed to obtain the products along with bio-energy (Fernand et al., 2017). Designing a sustainable bio-refinery for generation of sustainable food, fuels and chemicals poses great challenge and is largely influenced by a number of factors including biomass supply chain, techno-economic feasibility as well as the market dynamics (Konda et al., 2015). The main considerations for preferring macroalgal feedstock over terrestrial feedstock for bioenergy applications have been the higher photosynthetic efficiency (6–8%) than terrestrial plants (1.8–2.2%), faster growth, higher productivity, absence of lignin in their cell wall and greater ability to assimilate nutrients. Further, seaweed farming is done in the sea and, therefore, it does not
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compete with agricultural crops for land and water nor it requires agricultural inputs such as fertilizer and pesticides for their farming. The integrated bio-refinery approach has a number of issues to address; few of the most important are the production of high value products, least use of hazardous chemicals in the process and minimum waste disposal which have been well demonstrated by a number of studies (Baghel et al., 2015, Baghel et al., 2016; Trivedi et al., 2016). However, to date none of the reports have demonstrated an integrated biorefinery approach to recover marine macroalgal proteins together with the other established counterparts. Proteins are one of the most abundant biomolecules in a cell which are crucial for both, growth as well as regulation of various metabolic processes; this diversity of their functions is related to their structural complexity which makes them difficult to extract. Moreover, the ever expanding population of the world and the fluctuating economies demands a safe source which becomes proven to be the inexpensive as well as optimum in the required nutritional component so that the food scarcity and food security of a respective country be properly managed. The present study demonstrates an integrated process wherein protein is cogenerated along with other value added compounds such as mineral rich seaweed sap, total lipids, ulvan and cellulose from a green macroalgal species Ulva lactuca in a biorefinery model.
2. Materials and methods: Materials: All the chemicals used in the study were of analytical grade and purchased from Sigma-Aldrich (U.S.A.), solvents from Merck (Germany) and o-phthalaldehyde, sodium hydroxide, sodium tetraborate and sodium hypochlorite from Himedia laboratories (Mumbai, India). 2.1.
Sample collection:
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Ulva lactuca was collected from Veraval coast (N 20º 54.87'; E 70º 20.83'), Gujarat, India. The collection of fronds from one population was carried out to ascertain that they are derived from single mother plant. The biomass was collected along with sufficient seawater from the vicinity to avoid desiccation. The plants were transported to the laboratory immediately under dark conditions in a cool box with ice cool packs to avoid photo bleaching. Samples were washed several times with sea water to remove adhering sand, mud and other particles along with epiphytes. The sample was immediately frozen with liquid nitrogen and stored at -80˚C for analysis. 2.2.
Sap extraction:
The sap of the macroalgae contains valuable minerals which are essential for growth of agricultural crops. In the present study, an integrated process was developed such a way that most of the valuable macromolecules are successively extracted together with the sap extraction. To evaluate the effect of different sap extraction procedures on the mineral content, four different experiments were planned as follows: 2.2.1. Sap extraction by crushing the biomass (CB): In this method, 50 g of fresh alga was added with 100 ml of deionized water. It was crushed using kitchen mixer grinder up to the complete disintegration of thallus becomes visible. The alga mixture was filtered using a muslin cloth followed by 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to inductively coupled plasma atomic emission spectroscopy; ICP (PerkinElmer, Optima 2000, USA) analysis for mineral content determination. 2.2.2. Sap extraction from whole biomass (WB): In this method, 50 g of fresh alga was added with 100 ml of deionized water and kept at room temperature for 1 h. After the incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by
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filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.2.3. Sap extraction from whole biomass combined with heat treatment (WBH): In this method, 50 g of fresh alga was added with 100 ml of deionized water and incubated at 60 ˚C for 1 h. After the incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.2.4. Sap extraction from crushed biomass combined with heat treatment (CBH): In this method, 50 g of fresh alga was added with 100 ml of deionized water and crushed using a kitchen mixer grinder followed by incubation at 60 ˚C for 1 h. After incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.3.
Total lipid extraction: The residue remaining after sap extraction was subjected to total lipid extraction
following the method of (Bligh and Dyer, 1959). Briefly, the residue was mixed with chloroform and methanol in the ratio of 1:2. The sample was kept on a magnetic stirrer for at least 3 h to ensure complete lipid solubilisation. The mixture was transferred to a separating funnel followed by the addition of equal amount of distilled water. The funnel was allowed to stand until the two phases get separated evenly. The lower phase was collected and filtered through 0.21 µm filter paper and transferred to the vacuum evaporator to remove solvents. The total lipid was determined gravimetrically and processed for fatty acid determination (Kumari et al., 2011). A 0.5 g fresh weight of the residue was removed for dry weight
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calculation and elemental analysis using the instrument Elementar GmbH vario MICRO cube, calibrated using sulfanilamide as a reference standard. 2.4.
Ulvan extraction: The residue remaining after lipid extraction was subjected to ulvan extraction
(Jaulneau et al., 2010). Briefly, the process involves incubation of the residue in distilled water at 90 ˚C for 2 h. The mixture was allowed to cool up to it becomes easy to filter through muslin cloth and 0.21 µm filter paper. The filtered suspension was added with 3 volumes of iso-propanol and stirred vigorously for 30 minutes. The precipitates were recovered simply by cloth filtration and dried in oven and stored in an air tight container at room temperature. A 0.5 gram fresh weight of sample was removed for dry weight calculation and elemental analysis. 2.5.
Protein extraction: The residual biomass remaining after ulvan extraction was subjected to alkaline
treatment for protein extraction after fixing a standardized alkaline concentration (data not shown). The treatment involves use of 1N sodium hydroxide at 80 ˚C for complete dissolution of algal biomass such that proteins can be easily liberated into the system. The mixture was allowed to cool at room temperature and filtered using 0.21 µm filter followed by neutralization using 6N HCl. The suspension was dialyzed, lyophilized and stored in an air tight bottle at room temperature for analysis of total amino acids, heavy metals, and in-vitro digestibility assay. A 0.5 gram fresh weight of sample was removed for dry weight calculation and elemental analysis. 2.5.1. Amino acid analysis:
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The extracted proteins were subjected to amino acid analysis following previously published report with minor modifications (Kwanyuen and Burton, 2010). Briefly, the 10 mg of the extracted protein was subjected to acid hydrolysis using 500 µl of 6 N HCl at 110 °C for 24 h followed by drying under vaccum. For precolumn derivatization, the hydrolysed sample and amino acid standard mixture were neutralised using 20 µl of ethanol:water: triethylamine (TEA) in 2:2:1 (v/v), and dried under vacuum. The dried pellet was added with 20 µl of solvent mixture containing ethanol:water:TEA: Phenylisothiocyanate (PITC) in 7:1:1:1 (v/v) for derivatisation and were incubated at room temperature for 20 min in dark and dried under vaccum followed by solubilisation in 500 µl of 5 mM phosphate buffer, pH 7.4 with 5 % acetonitrile and filtered using 0.2 µm syringe filter to carry out high performance liquid chromatography (HPLC) analysis as described by Kwanyuen and Burton, 2010. 2.5.2. ICP analysis: Mineral composition of sap and heavy metal composition of protein was performed using ICP-OES analysis. The sap was filtered through 0.22 µm syringe filter prior to analysis and the 200 mg of extracted proteins was subjected to acid hydrolysis using 10 ml of concentrated nitric acid for overnight followed by addition of 2.5 ml concentrated perchloric acid and 250 µl sulfuric acid followed by heating until the complete cessation of white smoke from the sample. The digestion was added with 100 ml of 2 % HCl and filtered with 0.22 µm syringe filter followed by analysis using inductively coupled plasma atomic emission spectroscopy (PerkinElmer, Optima 2000, USA) (Santoso et al., 2006) 2.5.3. In vitro digestibility testing using OPA assay: The peptide bond hydrolysis of protein extracts was done using gastrointestinal enzymes such as pepsin and porcine pancreatin according to the manufacturer protocols. The 7
degree of hydrolysis of protein extract was measured using OPA assay (Wang et al., 2008). Briefly, the reagent was freshly prepared by combining 25 ml of 100 mM sodium tetraborate; 2.5 ml of 20% (w/w) SDS; 40 mg of OPA (solubilised in 1ml methanol); and 100 µl of βmercaptoethanol with final volume made up to 50 ml using MilliQ water. The 50 µl of hydrolysate was combined with 2 ml of OPA reagent mixture and allowed react for 4 minutes followed by measuring absorbance at 340 nm using glutathione as a standard. 2.6.
Cellulose extraction: The residues remaining after protein extraction was subjected to cellulose extraction.
The residue was added to 36% w/w sodium hypochlorite at pH 3 and incubated overnight at 65-70 ˚C. The sample was washed with distilled water up to it attains neutrality followed by the treatment with 0.5 M sodium hydroxide at 60 ˚C overnight. The residue was subjected to acid digestion using 5% HCl and heated up to the boiling followed by washing, up to neutrality. The residue was dried and stored at room temperature (Mihranyan et al., 2004).
3.
Results and Discussion:
3.1.
Sap extraction: The sap of marine macroalgae has been found to constitute several types of bioactives
including phytohormones and macro- and micro-elements of importance for plant growth and development (Prasad et al., 2010). The sap efficacy trials in field on various agronomically important crops has showed its effectiveness in improving yield as well as nutritional quality with reduction in recommended dose of chemical fertilizers (Singh et al., 2016; Sharma et al., 2017). All four sap extraction methods investigated in the present study have shown significant effect on the extraction and yielded sap with different concentration of both macro- and micro-elements (Table: 1). Among the methods tested, the sap extracted
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from WB method showed invariably lower elemental composition when compared with CBH and CB method. Although sap from CBH method yielded marginally increased yields of a majority of elements (Zn, Ca, K, Mg) examined in this study over CB method, it was not considered as the best first due to the fact that heating process accelerated the solubilisation of water soluble sulfated polysaccharides (ulvan) in addition to energy input. Therefore, the CB treatment where the biomass was mechanically disintegrated into tiny particles showed relatively comparable elemental composition with CBH and thus it was considered the best for sap extraction. The incubation of whole biomass in pure water (MilliQ water) lead to the release of macro- and micro- elements mostly due osmotic imbalance and thus may require huge amount of water for their complete extraction. 3.2.
Total lipid extraction: Lipids and their respective fatty acids; saturated fatty acids (SFA) without double
bonds in acyl chain, monounsaturated fatty acids (MUFA) with one double bond in acyl chain and polyunsaturated fatty acids (PUFA) with two or up to 6 double bonds in acyl chain are one of the crucial fundamental molecules for human nutrition. Humans are able to synthesize both SFAs and MUFAs but not PUFAs with the first bond on the third or sixth carbon atom thus, they have to be obtained from the diet containing the main sources such as chloroplasts of higher plants and fat of aquatic organisms (Mišurcová et al., 2011). The dietary pattern of humans has been altered by higher intake of total lipids with a high representation of saturated and trans-fatty acids which are detrimental to human health. The imbalance of ɷ3 and ɷ6 fatty acids leads to the progression of various pathophysiologies and therefore are considered as functional food and nutraceuticals with many health benefits. Although fish and fish oil are considered as the main source of PUFAs, especially docosahexaenoic acid (C22:6 ɷ3) and arachidonic acid (C20:4ɷ6) but the primary product of ɷ3 fatty acids in trophic chain for fish is a product of marine microorganisms and marine 9
algae (Mišurcová et al., 2011). Seaweeds have relatively low lipid contents, but the composition rich in C18 (linoleic and alpha-linolenic) fatty acids and low in C20 PUFAs; a combination that has been associated with the prevention of cardiovascular diseases, osteoarthritis and diabetes (Mišurcová et al., 2011). In the present study, palmitic acid found to be the most abundant among the total fatty acids detected in the biomass with 69.60 ± 21.36 % followed by eladic acid; 13.47 ± 8.14%, palmitoleic acid; 5.48 ± 3.02%, G-linoleic acid; 5.32 ± 3.58%, alpha-linoleic acid; 1.71 ± 1.24%, pentadecanoic acid; 1.04 ± 0.81%, docosanoic acid; 0.99 ± 1.23% and 5,8,11,14,17- eicosa pentanoic acid (EPA) with 0.26 ± 0.47%. However, besides higher saturated fatty acids contents, the biomass was reported with some essential fatty acids such as C18:2ɷ6 (linoleic acid) and C18:3ɷ3 (linolenic acid) and the eicosanoid precursors C20:5ɷ3 (eicosapentaenoic acid) with values falling under the recommended ratio of ɷ6/ɷ3 for prevention of chronic and cardiovascular diseases (WHO, 2003, Simopoulos, 2002). The subtle variations in fatty acid contents are attributable to both the environmental and genetic changes (Nelson et al., 2002). 3.3.
Ulvan extraction: Ulvan is the major water soluble sulfated polysaccharide found in Ulva lactuca and few
other Ulvaceae members. Due to its high water holding capacity it is readily soluble in water and it can be easily extracted just by using hot water extraction where heat effectively promotes its release from the tissue in to the extraction system. After the heat treatment followed by filtration; ulvan gets easily precipitated using iso-propanol. In the present study the final ulvan powder obtained was 19.90% by dry weight which is fairly in accordance with the previous reports (Robic et al., 2009). The ulvan obtained was characterized by Fourier Transform Infrared (FT-IR) Spectroscopy for the presence of characteristic functional groups and thermal gravimetric analysis for its thermal stability profile to support the assumption that up-stream processes did not affect considerably the ulvan yield from the macroalgal 10
biomass. The FT-IR spectrum of ulvan was consisted of absorption bands recorded at 1642 and 1448 cm−1 corresponding to asymmetrical and symmetrical stretching vibrations of the carboxylate group and at 1255 and 854 cm−1 for the stretching vibration of S=O and the bending vibration of C-O-S of sulphate groups in agreement with the previous reports confirming intact functional groups and their properties (Morelli and Chiellini, 2010). Thermogravimetric analysis is a simple and accurate method for studying the decomposition pattern and the thermal stability of biopolymers which occurs sequentially in four major steps that are desorption of physically absorbed water, removal of structural water, depolymerisation due to breakage of C–O and C–C bonds in the ring units resulting in the evolution of CO, CO2 and H2O followed by the formation of polynuclear aromatic and graphitic carbon structures. (Parikh and Madamwar, 2006) In the present study, ulvan follows a sequential thermal decomposition pattern with first acute drop in mass change with 33.77% at near to the 210 °C followed by the second major drop at 450 °C of about 15.40% and the last drop was with 4.83% at 690 °C leaving 41.96% residual mass. Several researchers have studied potential of ulvan as a dietary fibre and found that being the member of the cell wall polysaccharides, it is not degraded by human gastrointestinal enzymes (Lahaye and Robic, 2007) contributing to the water retention capacity of the fibres supporting its use as a bulking agent; helping in the prevention of pathologies related to intestinal transit dysfunctions (McPherson, 1993). Ulvan or derived oligosaccharides significantly lowered the level of serum total cholesterol, LDL-cholesterol, and reduced triglyceride, while they increased the levels of serum HDL-cholesterol, provide a preliminary evidence that supports its applicability to the design of functional foods and feed (Pengzhan et al., 2003a,b). The gastrointestinal and lipid metabolism benefits of ulvan are added with its immunomodulatory, antitumor (Kaeffer et al., 1999), strain-specific anti-influenza activities (Ivanova et al., 1994) and anticoagulant activities (Mao et al., 2006). 11
3.4.
Protein extraction: Protein extraction was the prime goal of the present study as it holds importance in
terms of both commercially valued product and as a forever demanded nutritional supplement. The protein contents of macroalgae is known to contain all essential amino acids (EAA) although seasonal variations in their concentrations are also reported (Galland-Irmouli et al., 1999). U. lactuca has showed a high protein content comparable to the traditional high protein plant sources justifying its direct use in human nutrition or for the development of balanced diets for animal nutrition (Ortiz et al., 2006). The protein content was traced as the mean of total protein content per gram dry weight (DW) biomass throughout the steps of the process to determine extraction efficiency and loss during the procedure. The inherent concentration of biomass was 16.18 ± 3.03% where at the end of the extraction, the lyophilized extracted protein was recorded as 11 ± 2.12 % DW biomass accounting for 68.75±4.01 % of the process efficiency. The results show condensation of protein content on each step as the progression of process (Fig.1). As illustrated in the figure, the total protein present in the initial biomass was 1.44 gm/gm DW biomass which attained 1.39 gm/gm DW upon sap extraction followed by 1.35 gm/gm DW biomass upon lipid extraction and 1.29 gm/gm DW biomass after ulvan extraction. Remarkably, the hot alkaline treatment liberated most of the protein content into the extraction system as evidenced by the residual total protein content of only 0.08 gm/gm DW after protein extraction. The residual basis calculations illustrated in the Fig. 1 and 2 reveals that there is a static balance of total protein throughout the procedure which is finally separated out with a minor remaining in the residue being subjected to cellulose extraction; approximately 5% total protein. This method provides greener approach towards protein extraction as traditionally it involves usage of phenol which is highly toxic and hazardous in nature. The present report is the first report presenting a non-
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hazardous and viable integrated approach for protein extraction targeted for food/feed applications. 3.4.1. Amino acid profile: The amino acid profile (Table: 2) favor its application in animal feed as it contents majority of the essential amino acids in the range of amino acid requirements of an adult according to the World Health Organisation (WHO) recommendations. The most abundant essential amino acid was iso-leucine; 23.857 ± 11.32 mg/gm protein, followed by histidine 22.920 ± 19.68, tyrosine; 12.710 ± 15.42, threonine 13.559 ± 2.65, phenyl alanine; 9.324 ± 13.19, leucine; 6.509 ± 2.37 and lysine; 1.450 ± 1.80 mg/gm protein whereas the valine, cysteine and methionine were not detected in the present system. The comparison between non-essential amino acid showed that arginine was the most abundant with 17.754 ± 3.02 mg/gm of the total protein followed by serine with 15.281 ± 15.89 mg/gm, aspartic acid with 14.423 ± 18.94 mg/gm, alanine 5.279 ± 7.47 mg/gm, glycine 1.076 ± 1.52 mg/gm and glutamic acid 0.321 ± 0.45 mg/gm whereas proline could not be detected in the present system. Comparing the total amino acid profile, iso-leucine was the highest with 16.51 ± 0.03% of total amino acids and glutamic acid was found to be lowest with 0.22 ± 0.24%. According to the WHO recommendations, iso-leucine, histidine, tyrosine, threonine, phenyl alanine contents from Ulva biomass are well comparative to the recommended values suggesting its potential role as a nutritional supplement. 3.4.2. ICP analysis: The recovered protein was also subjected to ICP analysis to analyze the presence of heavy metals and mineral contents such as arsenic, barium, boron, cadmium, chromium, copper, cobalt, iron, lead, lithium, nickel, manganese and zinc. As provided in the Table: 3, all of the analyzed heavy metals/ minerals are found to be far below the prescribed limits 13
provided by Food and Drug Administration (FDA). This supports the potential of ulva proteins as a safer alternative to protein supplements. 3.4.3. In-vitro digestibility: The assay method followed in this study is based on the reaction of o-phthalaldehyde (OPA) and β-mercaptoethanol with primary amines (Wang et al., 2008). The advantage of using this method is a single reagent serves both to inhibit proteolytic activities and to develop the reaction colour. Compared with other methods; Folin-Ciocalteau phenol reagent which is specific for tyrosine and tryptophan amino acids, exhibits interference from a large number of compounds. The casein was used as a digestion standard for determining the degree of hydrolysis and glutathione was used to make standard curve. Comparing the degree of hydrolysis, the lyophilized protein showed excellent digestibility; 85.86 ± 5.92% as compared with casein as a digestion standard. This can be attributed to the easy access of the mixtures of enzymes in the porcine pancreatin which act simultaneously on the protein substrate for hydrolysis due to the up-stream processing of the biomass that involves mechanical crushing followed by successive extractions ended with hot alkaline treatment that lead to the removal of the major macromolecular cellular contents from the biomass rendering it easily digestible through the digestive tract of animals and so increasing its bioavailability. However, it is recommended to extend the study for assessing its bioavailability as well as bio-accessibility to comply the digestibility results. Ulva lactuca has been studied previously for unravelling the potential of the proteins as a nutritional ingredient where it has been suggested that in addition to supplementing essential amino acids, the application may extend as an additional perspective in livestock production. Few reports have also showed evidence to support this finding which include, the application for enhancing health conditions of pigs fed with seaweeds as nutritional supplement
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(Gardiner et al., 2008; Dierick et al., 2010) and improvements in milk composition from algae fed dairy cows and ewes (Papadopoulos et al., 2002; Singh et al., 2003) which clearly demonstrates the potential of seaweeds as a good nutritional supplement for commercial animal farms. However, the extent of benefits available from seaweeds require more detailed analysis to regularise it as an essential ingredient of animal feed to ascertain any long term effects. The available studies render seaweeds as a comparable candidate for energy production with a higher economic value in future to decrease the increased pressure on the agricultural lands. 3.5.
Cellulose extraction: The cellulose extraction was the final step in this integrated approach as it is found to
be least affected by the up-stream treatments compared to any other components of the procedure which is evident from its prominent recovery of 10.35 ± 1.07 % on the dry weight basis and the FT-IR profile which is comparable to the previously available reports as the presence specific absorbance of C-H stretching at 2924 cm-1, bound water stretching at 1629 cm-1, C-H bending at 1427 cm-1 and C-O-C, C-O stretching in 1000-1200 cm-1 region corresponding to the presence of carbohydrates (Trivedi et al., 2016). After ulvan, cellulose is the major polysaccharide found in marine green macroalgae which has potential applications today for paper industries and as a feed stock of the rapidly emerging era of biofuels. Besides these two potential industrial applications, cellulose has been a preferred polymer for synthesis of nanocomposites (Oksman et al., 2016), Nano whiskers (Eichhorn, 2011), microcrystals (De Souza Lima and Borsali, 2004) and nanocrystals (Dong and Roman, 2007; Habibi et al., 2010) applied in varieties of research fields. The uses are not limited to the chemical research but have been rapidly expanded to biomedical applications, such as in regenerative medicines (Czaja et al., 2007). Yaich et al., 2015 reported that from the total insoluble dietary fibre contents of Ulva lactuca, cellulose and hemicellulose accounts for 15
49.08% suggesting that the high values of water holding capacity and oil holding capacity of the insoluble fibres was comparable to other commercial fibres, demonstrating its potential usefulness in the formulation of low calorie foods and in the stabilisation of foods rich in fat and emulsion. The biorefinery processes are advantageous if the intended biomolecules are present in abundance or the productivity of the source is higher. This phenomenon is recently reported and have provided a comparison between the related seaweed; Ulva ohnoi and a well-known source, soybean for their protein content as a function of growth rate as the average annual productivity of soybean is ~3 t ha−1 year−1 and the protein concentration of 40% resulting in productivity of 1.2 t ha−1 year−1 when compared with the fast growing landbased cultivation of U. ohnoi, shows that it can produce 18.4 t ha−1 year−1of protein (Mata et al., 2016). These findings mark green seaweeds as a potential candidate for biorefinery processes because despite the lower resident protein contents, its growth rate is so high that it can easily compensate the difference by its productivity. Macroalgae integration into a biorefinery is promising for its efficient conversion to value added products but there exists several challenges in feedstock production at competitive price, sustainable integrated design keeping in mind both industrial feasibility, socioeconomic and environmental concerns are some of the issues to be overcome for making the biorefinery as a successful marine enterprise. It has been assumed that zero waste processing technologies of macroalgae into food, chemicals and fuels will reduce the burden on the agriculture from arable land but it is a long way to realize this goal and also requires tremendous innovations in both upstream and downstream technologies.
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4. Conclusion: Macroalgae are increasingly gaining importance world over as a promising source for developing biorefinery for sustainable and effective utilisation of marine resource. The present study for the first time describes a method integrating protein recovery along with coproduction of other products from marine macroalgal feedstock. The protein digestibility as obtained from in vitro studies also confirms its suitability for its utilisation in food or feed supplements. The biorefinery approach reported in this study not only helps to realize greater value from feedstock but also provides ample opportunities for undertaking further value addition studies to develop newer products with newer applications opening newer markets. Acknowledgements: The authors gratefully acknowledge the DSIR, Ministry of Science and Technology, Govt. of India for financial support (DSIR/PACE/TDD/APPL/17/2013-14). We would like to thank reviewers as well as the handling editor for constructive comments. We are also grateful to the Director, CSIR-CSMCRI for encouragement and support, and the Central Analytical Instrumentation Facility for analytical support and services. The manuscript has CSIR-CSMCRI PRIS registration number PRIS 026/2017; Dated 16.02.2017. References: 1.
Baghel, R.S., Trivedi, N., Reddy, C.R.K., 2016. A simple process for recovery of a stream of products from marine macroalgal biomass. Bioresour. Technol. 203, 160–165.
2.
Baghel, R.S., Trivedi, N., Gupta, V., Neori, A., Reddy, C.R.K., Lali, A., Jha, B., 2015. Biorefining of marine macroalgal biomass for production of biofuel and commodity chemicals. Green Chem. 17, 2436–2443.
3.
Bixler, H.J., Porse, H., 2011. A decade of change in the seaweed hydrocolloids industry. J. Appl. Phycol. 23, 321–335.
4.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. 17
Can. J. Biochem. Biophysiol. 37, 911–915. 5.
Czaja, W.K., Young, D.J., Kawecki, M., Brown, R.M., 2007. The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 8, 1–12.
6.
De Souza Lima, M.M., Borsali, R., 2004. Rodlike cellulose microcrystals: Structure, properties, and applications. Macromol. Rapid Commun. 25, 771–787.
7.
Dierick, N., Ovyn, A., De Smet, S., 2010. In vitro assessment of the effect of intact marine brown macro-algae Ascophyllum nodosum on the gut flora of piglets. Livest. Sci. 133, 154– 156.
8.
Dong, S., Roman, M., 2007. Fluorescently labeled cellulose nanocrystals for bioimaging applications. J. Am. Chem. Soc. 129, 13810–13811.
9.
Eichhorn, S.J., 2011. Cellulose nanowhiskers: promising materials for advanced applications. Soft Matter 7, 303-315.
10. Fernand, F., Israel, A., Skjermo, J., Wichard, T., Timmermans, K.R., Golberg, A., 2017. Offshore macroalgae biomass for bioenergy production: Environmental aspects, technological achievements and challenges. Renew. Sustain. Energy Rev. 75, 35-45. 11. Galland-Irmouli, A.V., Fleurence, J., Lamghari, R., Lucon, M., Rouxel, C., Barbaroux, O., Bronowicki, J.P., Villaume, C., Gueant, J.L., 1999. Nutritional value of proteins from edible seaweed Palmaria palmata (Dulse). J. Nutr. Biochem. 10, 353–359. 12. Gardiner, G.E., Campbell, A.J., Doherty, J.V.O., Ross, R.P., Lawlor, P.G., 2008. Effect of Ascophyllum nodosum extract on growth performance , digestibility , carcass characteristics and selected intestinal microflora populations of grower – finisher pigs. Anim. Feed Sci. Tech. 141, 259–273. 13. Habibi, Y., Lucia, L.A., Rojas, O.J., 2010. Cellulose nanocrystals: Chemistry, self-assembly, and applications. Chem. Rev. 110, 3479–3500.
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14. Holdt, S.L., Kraan, S., 2011. Bioactive compounds in seaweed: Functional food applications and legislation. J. Appl. Phycol. 23, 543–597. 15. Ivanova, V., Rouseva, R., Kolarova, M., Serkedjieva, J., Rachev, R., Manolova, N., 1994. Isolation of a polysaccharide with antiviral effect from Ulva lactuca. Prep. Biochem. 24, 83– 97. 16. Jang, J.S., Cho, Y.K., Jeong, G.T., Kim, S.K., 2012. Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica. Bioprocess Biosyst. Eng. 35, 11–18. 17. Jaulneau, V., Lafitte, C., Jacquet, C., Fournier, S., Salamagne, S., Briand, X., EsquerréTugayé, M.T., Dumas, B. 2010. Ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway. J. Biomed. Biotechnol. 2010, 111 18. Kaeffer, B., Bénard, C., Lahaye, M., Blottière, H.M., Cherbut, C. 1999. Biological Properties of Ulvan, a New Source of Green Seaweed Sulfated Polysaccharides, on Cultured Normal and Cancerous Colonic Epithelial Cells. Planta Med. 65, 527–531. 19. Konda, N.V.S.N.M., Singh, S., Simmons, B.A., Klein-Marcuschamer, D., 2015. An Investigation on the Economic Feasibility of Macroalgae as a Potential Feedstock for Biorefineries. Bioenergy Res. 8, 1046–1056. 20. Kumari, P., Reddy, C.R.K., Jha, B., 2011. Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae. Anal. Biochem. 415, 134–144. 21. Kwanyuen, P., Burton, J.W., 2010. A Modified Amino Acid Analysis Using PITC Derivatization for Soybeans with Accurate Determination of Cysteine and Half-Cystine TL 87. J. Am. Oil Chem. Soc. 87, 127–132. 22. Lahaye, M., Robic, A., 2007. Structure and function properties of Ulvan, a polysaccharide from green seaweeds. Biomacromolecules 8, 1765–1774.
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23. Mao, W., Zang, X., Li, Y., Zhang, H., 2006. Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity. J. Appl. Phycol. 18, 9–14. 24. Mata, L., Magnusson, M., Paul, N.A., de Nys, R., 2016. The intensive land-based production of the green seaweeds Derbesia tenuissima and Ulva ohnoi: biomass and bioproducts. J. Appl. Phycol. 28, 365–375. 25. McPherson, R., 1993. Hand book of dietary fiber in human nutrition, in: G.A.S., S. (Ed.), CRC press, Boca Raton, FL, pp. 7–11. 26. Mihranyan, A., Llagostera, A.P., Karmhag, R., Strømme, M., Ek, R., 2004. Moisture sorption by cellulose powders of varying crystallinity. Int. J. Pharm. 269, 433–442. 27. Mišurcová, L., Ambrožová, J., Samek, D., 2011. Seaweed lipids as nutraceuticals. Adv. Food Nutr. Res. 64, 339–355. 28. Morelli, A., Chiellini, F., 2010. Ulvan as a new type of biomaterial from renewable resources: Functionalization and hydrogel preparation. Macromol. Chem. Phys. 211, 821– 832. 29. Nelson, M.M., Phleger, C.F., Nichols, P.D., 2002. Seasonal lipid composition in macroalgae of the northeastern Pacific Ocean. Bot. Mar. 45, 58–65. 30. Oksman, K., Aitomäki, Y., Mathew, A.P., Siqueira, G., Zhou, Q., Butylina, S., Tanpichai, S., Zhou, X., Hooshmand, S., 2016. Review of the recent developments in cellulose nanocomposite processing. Compos. Part A Appl. Sci. Manuf. 83, 2–18. 31. Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E., Osorio, A., Rios, A., 2006. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chem. 99, 98–104. 32. Papadopoulos, G., Goulas, C., Apostolaki, E., Abril, R., 2002. Effects of dietary supplements of algae, containing polyunsaturated fatty acids, on milk yield and the composition of milk products in dairy ewes. J. Dairy Res. 69, 357–365.
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33. Parikh, A., Madamwar, D., 2006. Partial characterization of extracellular polysaccharides from cyanobacteria. Bioresour. Technol. 97, 1822–1827. 34. Pengzhan, Y., Ning, L., Xiguang, L., Gefei, Z., Quanbin, Z., Pengcheng, L., 2003a. Antihyperlipidemic effects of different molecular weight sulfated polysaccharides from Ulva pertusa (Chlorophyta). Pharmacol. Res. 48, 543–549. 35. Pengzhan, Y., Quanbin, Z., Ning, L., Zuhong, X., Yanmei, W., Zhi'en, L., 2003b. Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity. J. Appl. Phycol. 15, 21–27. 36. Prasad, K., Das, A.K., Oza, M.D., Brahmbhatt, H., Siddhanta, A.K., Meena, R., Eswaran, K., Rajyaguru, M.R., Ghosh, P.K., 2010. Detection and quantification of some plant growth regulators in a seaweed-based foliar spray employing a mass spectrometric technique sans chromatographic separation. J. Agric. Food Chem. 58, 4594–4601. 37. Robic, A., Sassi, J.F., Dion, P., Lerat, Y., Lahaye, M., 2009. Seasonal variability of physicochemical and rheological properties of ulvan in two ulva species (chlorophyta) from the Brittany coast1. J. Phycol. 45, 962–973. 38. Simopoulos, A.P., 2002. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed. Pharmacother. 56, 365-379. 39. Santoso, J., Gunji, S., Yoshie-Stark, Y., Suzuki, T., 2006. Mineral Contents of Indonesian Seaweeds and Mineral Solubility Affected by Basic Cooking. Food Sci. Technol. Res. 12, 59–66. 40. Sharma, L., Banerjee, M., Malik, G.C., Vijay Anand, K. Sudhakar, G., Zodape T., Ghosh A., 2017. Sustainable agro-technology for enhancement of rice production in the red and lateritic soils using seaweed based biostimulants. J Clean Prod. 149, 968-975
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41. Singh, A.P., Avramis, C., Kramer, J.K.G., Marangoni, A.G., 2003. Algal meal supplementation of the cows’ diet alters the physical properties of milk fat. J. Dairy Res. 71, 66–73. 42. Singh, S., Singh M.K., Pal, S.K., Trivedi, K., Yesuraj, D., Singh, C.S., Vijay Anand, K.G, chandramohan, M., Patidar, R., Kubavat, D., Zodape, S.T., Ghosh, A., 2016. Sustainable enhancement in yield and quality of rain-fed maize through Gracilaria edulis and Kappaphycus alvarezii seaweed sap. J Appl Phycol. 28, 2099-2112. 43. Trivedi, N., Baghel, R.S., Bothwell, J., Gupta, V., Reddy, C.R.K., Lali, A.M., Jha, B., 2016. An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass. Sci. Rep. 6, 30728. 44. Wang, D., Wang, L. jun, Zhu, F. xue, Zhu, J. ye, Chen, X.D., Zou, L., Saito, M., Li, L. te, 2008. In vitro and in vivo studies on the antioxidant activities of the aqueous extracts of Douchi (a traditional Chinese salt-fermented soybean food). Food Chem. 107, 1421–1428. 45. WHO. 2003. Diet, nutrition and the prevention of chronic diseases. WHO technical report series 916. Geneva: WHO. 46. Yaich, H., Garna, H., Bchir, B., Besbes, S., Paquot, M., Richel, A., Blecker, C., Attia, H., 2015. Chemical composition and functional properties of dietary fibre extracted by Englyst and Prosky methods from the alga Ulva lactuca collected in Tunisia. Algal Res. 9, 65–73. Figures captions: Figure 1 Data on protein content at different biorefinery stages Figure 2 Data on protein recovery efficiency Vs biomass consumption at different stages of biorefinery
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Figures
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Tables
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Highlights • • •
Biorefinery approach for integrated extraction of proteins from marine macroalgae The digestibility of protein is more than 85% based on in vitro enzyme hydrolysis Sap extraction was optimized for effective recovery of constituents from biomass
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S0960-8524(17)31053-2 http://dx.doi.org/10.1016/j.biortech.2017.06.149 BITE 18386
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
29 May 2017 24 June 2017 26 June 2017
Please cite this article as: Gajaria, T.K., Suthar, P., Baghel, R.S., B.Balar, N., Sharnagat, P., Mantri, V.A., Reddy, C.R.K., Integration of protein extraction with a stream of byproducts from marine macroalgae: a model forms the basis for marine bioeconomy, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech. 2017.06.149
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Integration of protein extraction with a stream of byproducts from marine macroalgae: a model forms the basis for marine bioeconomy Tejal K. Gajaria1,2, Poornima Suthar1, Ravi S. Baghel1,2, Nikunj B.Balar1,2, Preeti Sharnagat1, Vaibhav A. Mantri1,2 and C.R.K. Reddy1,2 *
1
Division of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine
Chemicals Research Institute, Bhavnagar-364002, India 2
Academy of Scientific and Innovative Research (AcSIR), New Delhi, India
Abstract The present study describes an advanced biorefinery model for marine macroalgae that assumes significant importance in the context of marine bio-economy. The method investigated in this study integrates the extraction of crude proteins with recovery of minerals rich sap, lipids, ulvan and cellulose from fresh biomass of Ulva lactuca. The protein content extracted was 11±2.12 % on dry weight basis with recovery efficiency of 68.75±4.01%. The amino acid composition of crude protein fraction showed isoleucine as the most abundant amino acid with 16.51±0.03% followed by histidine, arginine, tyrosine, serine, aspartic acid, threonine, phenyl alanine, leucine, alanine, lysine, glycine and glutamic acid (0.22±0.24%). The digestibility of protein was as high as 85.86±5.92% indicating its suitability for use in food supplements. The protein production with co-recovery of other products would not only result in effective utilisation marine macroalgal resources but also forms the basis for marine bioeconomy.
Keywords: Algal salt, Integrated biorefinery, Nutritional supplement, Sustainable process, Ulvan *Author for correspondence Tel: +91 278 257 0885 Fax: + 91 278 256 6970 / 256 7562 Email: [email protected]
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1. Introduction Marine macroalgae, commonly known as seaweeds, comprise of assemblage of diverse autotrophic lower plants that are not only integral part of oceanic ecosystem but also play an important role in CO2 sequestration besides rendering invaluable ecological services. Seaweeds have historically been used as part of human diets in some Fareast countries (Holdt and Kraan, 2011) while in other parts of the world they are used as a source of raw material for extraction of industrially import hydrocolloids such as agar, carrageenan and alginates (Bixler and Porse, 2011). In recent times, seaweeds have also been gaining considerable importance globally as non-lignocellulosic feedstock for production of bioenergy (Jang et al., 2012). The macroalgal biorefinery is one of the rapidly emerging frontier research areas of bio-processing for a sustainable production of variety of biomolecules. It possesses immense potential to provide possible alternatives to both global environmental concerns and a noncompetitive green source for production of nutrient supplements as well as other commodity materials. Bio-refinery utilises biomass components that are synthesised as a function of complex photosynthesis and therefore they have to be effectively processed to obtain the products along with bio-energy (Fernand et al., 2017). Designing a sustainable bio-refinery for generation of sustainable food, fuels and chemicals poses great challenge and is largely influenced by a number of factors including biomass supply chain, techno-economic feasibility as well as the market dynamics (Konda et al., 2015). The main considerations for preferring macroalgal feedstock over terrestrial feedstock for bioenergy applications have been the higher photosynthetic efficiency (6–8%) than terrestrial plants (1.8–2.2%), faster growth, higher productivity, absence of lignin in their cell wall and greater ability to assimilate nutrients. Further, seaweed farming is done in the sea and, therefore, it does not
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compete with agricultural crops for land and water nor it requires agricultural inputs such as fertilizer and pesticides for their farming. The integrated bio-refinery approach has a number of issues to address; few of the most important are the production of high value products, least use of hazardous chemicals in the process and minimum waste disposal which have been well demonstrated by a number of studies (Baghel et al., 2015, Baghel et al., 2016; Trivedi et al., 2016). However, to date none of the reports have demonstrated an integrated biorefinery approach to recover marine macroalgal proteins together with the other established counterparts. Proteins are one of the most abundant biomolecules in a cell which are crucial for both, growth as well as regulation of various metabolic processes; this diversity of their functions is related to their structural complexity which makes them difficult to extract. Moreover, the ever expanding population of the world and the fluctuating economies demands a safe source which becomes proven to be the inexpensive as well as optimum in the required nutritional component so that the food scarcity and food security of a respective country be properly managed. The present study demonstrates an integrated process wherein protein is cogenerated along with other value added compounds such as mineral rich seaweed sap, total lipids, ulvan and cellulose from a green macroalgal species Ulva lactuca in a biorefinery model.
2. Materials and methods: Materials: All the chemicals used in the study were of analytical grade and purchased from Sigma-Aldrich (U.S.A.), solvents from Merck (Germany) and o-phthalaldehyde, sodium hydroxide, sodium tetraborate and sodium hypochlorite from Himedia laboratories (Mumbai, India). 2.1.
Sample collection:
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Ulva lactuca was collected from Veraval coast (N 20º 54.87'; E 70º 20.83'), Gujarat, India. The collection of fronds from one population was carried out to ascertain that they are derived from single mother plant. The biomass was collected along with sufficient seawater from the vicinity to avoid desiccation. The plants were transported to the laboratory immediately under dark conditions in a cool box with ice cool packs to avoid photo bleaching. Samples were washed several times with sea water to remove adhering sand, mud and other particles along with epiphytes. The sample was immediately frozen with liquid nitrogen and stored at -80˚C for analysis. 2.2.
Sap extraction:
The sap of the macroalgae contains valuable minerals which are essential for growth of agricultural crops. In the present study, an integrated process was developed such a way that most of the valuable macromolecules are successively extracted together with the sap extraction. To evaluate the effect of different sap extraction procedures on the mineral content, four different experiments were planned as follows: 2.2.1. Sap extraction by crushing the biomass (CB): In this method, 50 g of fresh alga was added with 100 ml of deionized water. It was crushed using kitchen mixer grinder up to the complete disintegration of thallus becomes visible. The alga mixture was filtered using a muslin cloth followed by 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to inductively coupled plasma atomic emission spectroscopy; ICP (PerkinElmer, Optima 2000, USA) analysis for mineral content determination. 2.2.2. Sap extraction from whole biomass (WB): In this method, 50 g of fresh alga was added with 100 ml of deionized water and kept at room temperature for 1 h. After the incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by
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filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.2.3. Sap extraction from whole biomass combined with heat treatment (WBH): In this method, 50 g of fresh alga was added with 100 ml of deionized water and incubated at 60 ˚C for 1 h. After the incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.2.4. Sap extraction from crushed biomass combined with heat treatment (CBH): In this method, 50 g of fresh alga was added with 100 ml of deionized water and crushed using a kitchen mixer grinder followed by incubation at 60 ˚C for 1 h. After incubation, the sap was collected by squeezing the biomass using a muslin cloth followed by filtration using 0.21µm filter paper. The sap was stored at -20 ˚C and subjected to ICP analysis for mineral content determination. 2.3.
Total lipid extraction: The residue remaining after sap extraction was subjected to total lipid extraction
following the method of (Bligh and Dyer, 1959). Briefly, the residue was mixed with chloroform and methanol in the ratio of 1:2. The sample was kept on a magnetic stirrer for at least 3 h to ensure complete lipid solubilisation. The mixture was transferred to a separating funnel followed by the addition of equal amount of distilled water. The funnel was allowed to stand until the two phases get separated evenly. The lower phase was collected and filtered through 0.21 µm filter paper and transferred to the vacuum evaporator to remove solvents. The total lipid was determined gravimetrically and processed for fatty acid determination (Kumari et al., 2011). A 0.5 g fresh weight of the residue was removed for dry weight
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calculation and elemental analysis using the instrument Elementar GmbH vario MICRO cube, calibrated using sulfanilamide as a reference standard. 2.4.
Ulvan extraction: The residue remaining after lipid extraction was subjected to ulvan extraction
(Jaulneau et al., 2010). Briefly, the process involves incubation of the residue in distilled water at 90 ˚C for 2 h. The mixture was allowed to cool up to it becomes easy to filter through muslin cloth and 0.21 µm filter paper. The filtered suspension was added with 3 volumes of iso-propanol and stirred vigorously for 30 minutes. The precipitates were recovered simply by cloth filtration and dried in oven and stored in an air tight container at room temperature. A 0.5 gram fresh weight of sample was removed for dry weight calculation and elemental analysis. 2.5.
Protein extraction: The residual biomass remaining after ulvan extraction was subjected to alkaline
treatment for protein extraction after fixing a standardized alkaline concentration (data not shown). The treatment involves use of 1N sodium hydroxide at 80 ˚C for complete dissolution of algal biomass such that proteins can be easily liberated into the system. The mixture was allowed to cool at room temperature and filtered using 0.21 µm filter followed by neutralization using 6N HCl. The suspension was dialyzed, lyophilized and stored in an air tight bottle at room temperature for analysis of total amino acids, heavy metals, and in-vitro digestibility assay. A 0.5 gram fresh weight of sample was removed for dry weight calculation and elemental analysis. 2.5.1. Amino acid analysis:
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The extracted proteins were subjected to amino acid analysis following previously published report with minor modifications (Kwanyuen and Burton, 2010). Briefly, the 10 mg of the extracted protein was subjected to acid hydrolysis using 500 µl of 6 N HCl at 110 °C for 24 h followed by drying under vaccum. For precolumn derivatization, the hydrolysed sample and amino acid standard mixture were neutralised using 20 µl of ethanol:water: triethylamine (TEA) in 2:2:1 (v/v), and dried under vacuum. The dried pellet was added with 20 µl of solvent mixture containing ethanol:water:TEA: Phenylisothiocyanate (PITC) in 7:1:1:1 (v/v) for derivatisation and were incubated at room temperature for 20 min in dark and dried under vaccum followed by solubilisation in 500 µl of 5 mM phosphate buffer, pH 7.4 with 5 % acetonitrile and filtered using 0.2 µm syringe filter to carry out high performance liquid chromatography (HPLC) analysis as described by Kwanyuen and Burton, 2010. 2.5.2. ICP analysis: Mineral composition of sap and heavy metal composition of protein was performed using ICP-OES analysis. The sap was filtered through 0.22 µm syringe filter prior to analysis and the 200 mg of extracted proteins was subjected to acid hydrolysis using 10 ml of concentrated nitric acid for overnight followed by addition of 2.5 ml concentrated perchloric acid and 250 µl sulfuric acid followed by heating until the complete cessation of white smoke from the sample. The digestion was added with 100 ml of 2 % HCl and filtered with 0.22 µm syringe filter followed by analysis using inductively coupled plasma atomic emission spectroscopy (PerkinElmer, Optima 2000, USA) (Santoso et al., 2006) 2.5.3. In vitro digestibility testing using OPA assay: The peptide bond hydrolysis of protein extracts was done using gastrointestinal enzymes such as pepsin and porcine pancreatin according to the manufacturer protocols. The 7
degree of hydrolysis of protein extract was measured using OPA assay (Wang et al., 2008). Briefly, the reagent was freshly prepared by combining 25 ml of 100 mM sodium tetraborate; 2.5 ml of 20% (w/w) SDS; 40 mg of OPA (solubilised in 1ml methanol); and 100 µl of βmercaptoethanol with final volume made up to 50 ml using MilliQ water. The 50 µl of hydrolysate was combined with 2 ml of OPA reagent mixture and allowed react for 4 minutes followed by measuring absorbance at 340 nm using glutathione as a standard. 2.6.
Cellulose extraction: The residues remaining after protein extraction was subjected to cellulose extraction.
The residue was added to 36% w/w sodium hypochlorite at pH 3 and incubated overnight at 65-70 ˚C. The sample was washed with distilled water up to it attains neutrality followed by the treatment with 0.5 M sodium hydroxide at 60 ˚C overnight. The residue was subjected to acid digestion using 5% HCl and heated up to the boiling followed by washing, up to neutrality. The residue was dried and stored at room temperature (Mihranyan et al., 2004).
3.
Results and Discussion:
3.1.
Sap extraction: The sap of marine macroalgae has been found to constitute several types of bioactives
including phytohormones and macro- and micro-elements of importance for plant growth and development (Prasad et al., 2010). The sap efficacy trials in field on various agronomically important crops has showed its effectiveness in improving yield as well as nutritional quality with reduction in recommended dose of chemical fertilizers (Singh et al., 2016; Sharma et al., 2017). All four sap extraction methods investigated in the present study have shown significant effect on the extraction and yielded sap with different concentration of both macro- and micro-elements (Table: 1). Among the methods tested, the sap extracted
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from WB method showed invariably lower elemental composition when compared with CBH and CB method. Although sap from CBH method yielded marginally increased yields of a majority of elements (Zn, Ca, K, Mg) examined in this study over CB method, it was not considered as the best first due to the fact that heating process accelerated the solubilisation of water soluble sulfated polysaccharides (ulvan) in addition to energy input. Therefore, the CB treatment where the biomass was mechanically disintegrated into tiny particles showed relatively comparable elemental composition with CBH and thus it was considered the best for sap extraction. The incubation of whole biomass in pure water (MilliQ water) lead to the release of macro- and micro- elements mostly due osmotic imbalance and thus may require huge amount of water for their complete extraction. 3.2.
Total lipid extraction: Lipids and their respective fatty acids; saturated fatty acids (SFA) without double
bonds in acyl chain, monounsaturated fatty acids (MUFA) with one double bond in acyl chain and polyunsaturated fatty acids (PUFA) with two or up to 6 double bonds in acyl chain are one of the crucial fundamental molecules for human nutrition. Humans are able to synthesize both SFAs and MUFAs but not PUFAs with the first bond on the third or sixth carbon atom thus, they have to be obtained from the diet containing the main sources such as chloroplasts of higher plants and fat of aquatic organisms (Mišurcová et al., 2011). The dietary pattern of humans has been altered by higher intake of total lipids with a high representation of saturated and trans-fatty acids which are detrimental to human health. The imbalance of ɷ3 and ɷ6 fatty acids leads to the progression of various pathophysiologies and therefore are considered as functional food and nutraceuticals with many health benefits. Although fish and fish oil are considered as the main source of PUFAs, especially docosahexaenoic acid (C22:6 ɷ3) and arachidonic acid (C20:4ɷ6) but the primary product of ɷ3 fatty acids in trophic chain for fish is a product of marine microorganisms and marine 9
algae (Mišurcová et al., 2011). Seaweeds have relatively low lipid contents, but the composition rich in C18 (linoleic and alpha-linolenic) fatty acids and low in C20 PUFAs; a combination that has been associated with the prevention of cardiovascular diseases, osteoarthritis and diabetes (Mišurcová et al., 2011). In the present study, palmitic acid found to be the most abundant among the total fatty acids detected in the biomass with 69.60 ± 21.36 % followed by eladic acid; 13.47 ± 8.14%, palmitoleic acid; 5.48 ± 3.02%, G-linoleic acid; 5.32 ± 3.58%, alpha-linoleic acid; 1.71 ± 1.24%, pentadecanoic acid; 1.04 ± 0.81%, docosanoic acid; 0.99 ± 1.23% and 5,8,11,14,17- eicosa pentanoic acid (EPA) with 0.26 ± 0.47%. However, besides higher saturated fatty acids contents, the biomass was reported with some essential fatty acids such as C18:2ɷ6 (linoleic acid) and C18:3ɷ3 (linolenic acid) and the eicosanoid precursors C20:5ɷ3 (eicosapentaenoic acid) with values falling under the recommended ratio of ɷ6/ɷ3 for prevention of chronic and cardiovascular diseases (WHO, 2003, Simopoulos, 2002). The subtle variations in fatty acid contents are attributable to both the environmental and genetic changes (Nelson et al., 2002). 3.3.
Ulvan extraction: Ulvan is the major water soluble sulfated polysaccharide found in Ulva lactuca and few
other Ulvaceae members. Due to its high water holding capacity it is readily soluble in water and it can be easily extracted just by using hot water extraction where heat effectively promotes its release from the tissue in to the extraction system. After the heat treatment followed by filtration; ulvan gets easily precipitated using iso-propanol. In the present study the final ulvan powder obtained was 19.90% by dry weight which is fairly in accordance with the previous reports (Robic et al., 2009). The ulvan obtained was characterized by Fourier Transform Infrared (FT-IR) Spectroscopy for the presence of characteristic functional groups and thermal gravimetric analysis for its thermal stability profile to support the assumption that up-stream processes did not affect considerably the ulvan yield from the macroalgal 10
biomass. The FT-IR spectrum of ulvan was consisted of absorption bands recorded at 1642 and 1448 cm−1 corresponding to asymmetrical and symmetrical stretching vibrations of the carboxylate group and at 1255 and 854 cm−1 for the stretching vibration of S=O and the bending vibration of C-O-S of sulphate groups in agreement with the previous reports confirming intact functional groups and their properties (Morelli and Chiellini, 2010). Thermogravimetric analysis is a simple and accurate method for studying the decomposition pattern and the thermal stability of biopolymers which occurs sequentially in four major steps that are desorption of physically absorbed water, removal of structural water, depolymerisation due to breakage of C–O and C–C bonds in the ring units resulting in the evolution of CO, CO2 and H2O followed by the formation of polynuclear aromatic and graphitic carbon structures. (Parikh and Madamwar, 2006) In the present study, ulvan follows a sequential thermal decomposition pattern with first acute drop in mass change with 33.77% at near to the 210 °C followed by the second major drop at 450 °C of about 15.40% and the last drop was with 4.83% at 690 °C leaving 41.96% residual mass. Several researchers have studied potential of ulvan as a dietary fibre and found that being the member of the cell wall polysaccharides, it is not degraded by human gastrointestinal enzymes (Lahaye and Robic, 2007) contributing to the water retention capacity of the fibres supporting its use as a bulking agent; helping in the prevention of pathologies related to intestinal transit dysfunctions (McPherson, 1993). Ulvan or derived oligosaccharides significantly lowered the level of serum total cholesterol, LDL-cholesterol, and reduced triglyceride, while they increased the levels of serum HDL-cholesterol, provide a preliminary evidence that supports its applicability to the design of functional foods and feed (Pengzhan et al., 2003a,b). The gastrointestinal and lipid metabolism benefits of ulvan are added with its immunomodulatory, antitumor (Kaeffer et al., 1999), strain-specific anti-influenza activities (Ivanova et al., 1994) and anticoagulant activities (Mao et al., 2006). 11
3.4.
Protein extraction: Protein extraction was the prime goal of the present study as it holds importance in
terms of both commercially valued product and as a forever demanded nutritional supplement. The protein contents of macroalgae is known to contain all essential amino acids (EAA) although seasonal variations in their concentrations are also reported (Galland-Irmouli et al., 1999). U. lactuca has showed a high protein content comparable to the traditional high protein plant sources justifying its direct use in human nutrition or for the development of balanced diets for animal nutrition (Ortiz et al., 2006). The protein content was traced as the mean of total protein content per gram dry weight (DW) biomass throughout the steps of the process to determine extraction efficiency and loss during the procedure. The inherent concentration of biomass was 16.18 ± 3.03% where at the end of the extraction, the lyophilized extracted protein was recorded as 11 ± 2.12 % DW biomass accounting for 68.75±4.01 % of the process efficiency. The results show condensation of protein content on each step as the progression of process (Fig.1). As illustrated in the figure, the total protein present in the initial biomass was 1.44 gm/gm DW biomass which attained 1.39 gm/gm DW upon sap extraction followed by 1.35 gm/gm DW biomass upon lipid extraction and 1.29 gm/gm DW biomass after ulvan extraction. Remarkably, the hot alkaline treatment liberated most of the protein content into the extraction system as evidenced by the residual total protein content of only 0.08 gm/gm DW after protein extraction. The residual basis calculations illustrated in the Fig. 1 and 2 reveals that there is a static balance of total protein throughout the procedure which is finally separated out with a minor remaining in the residue being subjected to cellulose extraction; approximately 5% total protein. This method provides greener approach towards protein extraction as traditionally it involves usage of phenol which is highly toxic and hazardous in nature. The present report is the first report presenting a non-
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hazardous and viable integrated approach for protein extraction targeted for food/feed applications. 3.4.1. Amino acid profile: The amino acid profile (Table: 2) favor its application in animal feed as it contents majority of the essential amino acids in the range of amino acid requirements of an adult according to the World Health Organisation (WHO) recommendations. The most abundant essential amino acid was iso-leucine; 23.857 ± 11.32 mg/gm protein, followed by histidine 22.920 ± 19.68, tyrosine; 12.710 ± 15.42, threonine 13.559 ± 2.65, phenyl alanine; 9.324 ± 13.19, leucine; 6.509 ± 2.37 and lysine; 1.450 ± 1.80 mg/gm protein whereas the valine, cysteine and methionine were not detected in the present system. The comparison between non-essential amino acid showed that arginine was the most abundant with 17.754 ± 3.02 mg/gm of the total protein followed by serine with 15.281 ± 15.89 mg/gm, aspartic acid with 14.423 ± 18.94 mg/gm, alanine 5.279 ± 7.47 mg/gm, glycine 1.076 ± 1.52 mg/gm and glutamic acid 0.321 ± 0.45 mg/gm whereas proline could not be detected in the present system. Comparing the total amino acid profile, iso-leucine was the highest with 16.51 ± 0.03% of total amino acids and glutamic acid was found to be lowest with 0.22 ± 0.24%. According to the WHO recommendations, iso-leucine, histidine, tyrosine, threonine, phenyl alanine contents from Ulva biomass are well comparative to the recommended values suggesting its potential role as a nutritional supplement. 3.4.2. ICP analysis: The recovered protein was also subjected to ICP analysis to analyze the presence of heavy metals and mineral contents such as arsenic, barium, boron, cadmium, chromium, copper, cobalt, iron, lead, lithium, nickel, manganese and zinc. As provided in the Table: 3, all of the analyzed heavy metals/ minerals are found to be far below the prescribed limits 13
provided by Food and Drug Administration (FDA). This supports the potential of ulva proteins as a safer alternative to protein supplements. 3.4.3. In-vitro digestibility: The assay method followed in this study is based on the reaction of o-phthalaldehyde (OPA) and β-mercaptoethanol with primary amines (Wang et al., 2008). The advantage of using this method is a single reagent serves both to inhibit proteolytic activities and to develop the reaction colour. Compared with other methods; Folin-Ciocalteau phenol reagent which is specific for tyrosine and tryptophan amino acids, exhibits interference from a large number of compounds. The casein was used as a digestion standard for determining the degree of hydrolysis and glutathione was used to make standard curve. Comparing the degree of hydrolysis, the lyophilized protein showed excellent digestibility; 85.86 ± 5.92% as compared with casein as a digestion standard. This can be attributed to the easy access of the mixtures of enzymes in the porcine pancreatin which act simultaneously on the protein substrate for hydrolysis due to the up-stream processing of the biomass that involves mechanical crushing followed by successive extractions ended with hot alkaline treatment that lead to the removal of the major macromolecular cellular contents from the biomass rendering it easily digestible through the digestive tract of animals and so increasing its bioavailability. However, it is recommended to extend the study for assessing its bioavailability as well as bio-accessibility to comply the digestibility results. Ulva lactuca has been studied previously for unravelling the potential of the proteins as a nutritional ingredient where it has been suggested that in addition to supplementing essential amino acids, the application may extend as an additional perspective in livestock production. Few reports have also showed evidence to support this finding which include, the application for enhancing health conditions of pigs fed with seaweeds as nutritional supplement
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(Gardiner et al., 2008; Dierick et al., 2010) and improvements in milk composition from algae fed dairy cows and ewes (Papadopoulos et al., 2002; Singh et al., 2003) which clearly demonstrates the potential of seaweeds as a good nutritional supplement for commercial animal farms. However, the extent of benefits available from seaweeds require more detailed analysis to regularise it as an essential ingredient of animal feed to ascertain any long term effects. The available studies render seaweeds as a comparable candidate for energy production with a higher economic value in future to decrease the increased pressure on the agricultural lands. 3.5.
Cellulose extraction: The cellulose extraction was the final step in this integrated approach as it is found to
be least affected by the up-stream treatments compared to any other components of the procedure which is evident from its prominent recovery of 10.35 ± 1.07 % on the dry weight basis and the FT-IR profile which is comparable to the previously available reports as the presence specific absorbance of C-H stretching at 2924 cm-1, bound water stretching at 1629 cm-1, C-H bending at 1427 cm-1 and C-O-C, C-O stretching in 1000-1200 cm-1 region corresponding to the presence of carbohydrates (Trivedi et al., 2016). After ulvan, cellulose is the major polysaccharide found in marine green macroalgae which has potential applications today for paper industries and as a feed stock of the rapidly emerging era of biofuels. Besides these two potential industrial applications, cellulose has been a preferred polymer for synthesis of nanocomposites (Oksman et al., 2016), Nano whiskers (Eichhorn, 2011), microcrystals (De Souza Lima and Borsali, 2004) and nanocrystals (Dong and Roman, 2007; Habibi et al., 2010) applied in varieties of research fields. The uses are not limited to the chemical research but have been rapidly expanded to biomedical applications, such as in regenerative medicines (Czaja et al., 2007). Yaich et al., 2015 reported that from the total insoluble dietary fibre contents of Ulva lactuca, cellulose and hemicellulose accounts for 15
49.08% suggesting that the high values of water holding capacity and oil holding capacity of the insoluble fibres was comparable to other commercial fibres, demonstrating its potential usefulness in the formulation of low calorie foods and in the stabilisation of foods rich in fat and emulsion. The biorefinery processes are advantageous if the intended biomolecules are present in abundance or the productivity of the source is higher. This phenomenon is recently reported and have provided a comparison between the related seaweed; Ulva ohnoi and a well-known source, soybean for their protein content as a function of growth rate as the average annual productivity of soybean is ~3 t ha−1 year−1 and the protein concentration of 40% resulting in productivity of 1.2 t ha−1 year−1 when compared with the fast growing landbased cultivation of U. ohnoi, shows that it can produce 18.4 t ha−1 year−1of protein (Mata et al., 2016). These findings mark green seaweeds as a potential candidate for biorefinery processes because despite the lower resident protein contents, its growth rate is so high that it can easily compensate the difference by its productivity. Macroalgae integration into a biorefinery is promising for its efficient conversion to value added products but there exists several challenges in feedstock production at competitive price, sustainable integrated design keeping in mind both industrial feasibility, socioeconomic and environmental concerns are some of the issues to be overcome for making the biorefinery as a successful marine enterprise. It has been assumed that zero waste processing technologies of macroalgae into food, chemicals and fuels will reduce the burden on the agriculture from arable land but it is a long way to realize this goal and also requires tremendous innovations in both upstream and downstream technologies.
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4. Conclusion: Macroalgae are increasingly gaining importance world over as a promising source for developing biorefinery for sustainable and effective utilisation of marine resource. The present study for the first time describes a method integrating protein recovery along with coproduction of other products from marine macroalgal feedstock. The protein digestibility as obtained from in vitro studies also confirms its suitability for its utilisation in food or feed supplements. The biorefinery approach reported in this study not only helps to realize greater value from feedstock but also provides ample opportunities for undertaking further value addition studies to develop newer products with newer applications opening newer markets. Acknowledgements: The authors gratefully acknowledge the DSIR, Ministry of Science and Technology, Govt. of India for financial support (DSIR/PACE/TDD/APPL/17/2013-14). We would like to thank reviewers as well as the handling editor for constructive comments. We are also grateful to the Director, CSIR-CSMCRI for encouragement and support, and the Central Analytical Instrumentation Facility for analytical support and services. The manuscript has CSIR-CSMCRI PRIS registration number PRIS 026/2017; Dated 16.02.2017. References: 1.
Baghel, R.S., Trivedi, N., Reddy, C.R.K., 2016. A simple process for recovery of a stream of products from marine macroalgal biomass. Bioresour. Technol. 203, 160–165.
2.
Baghel, R.S., Trivedi, N., Gupta, V., Neori, A., Reddy, C.R.K., Lali, A., Jha, B., 2015. Biorefining of marine macroalgal biomass for production of biofuel and commodity chemicals. Green Chem. 17, 2436–2443.
3.
Bixler, H.J., Porse, H., 2011. A decade of change in the seaweed hydrocolloids industry. J. Appl. Phycol. 23, 321–335.
4.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. 17
Can. J. Biochem. Biophysiol. 37, 911–915. 5.
Czaja, W.K., Young, D.J., Kawecki, M., Brown, R.M., 2007. The future prospects of microbial cellulose in biomedical applications. Biomacromolecules 8, 1–12.
6.
De Souza Lima, M.M., Borsali, R., 2004. Rodlike cellulose microcrystals: Structure, properties, and applications. Macromol. Rapid Commun. 25, 771–787.
7.
Dierick, N., Ovyn, A., De Smet, S., 2010. In vitro assessment of the effect of intact marine brown macro-algae Ascophyllum nodosum on the gut flora of piglets. Livest. Sci. 133, 154– 156.
8.
Dong, S., Roman, M., 2007. Fluorescently labeled cellulose nanocrystals for bioimaging applications. J. Am. Chem. Soc. 129, 13810–13811.
9.
Eichhorn, S.J., 2011. Cellulose nanowhiskers: promising materials for advanced applications. Soft Matter 7, 303-315.
10. Fernand, F., Israel, A., Skjermo, J., Wichard, T., Timmermans, K.R., Golberg, A., 2017. Offshore macroalgae biomass for bioenergy production: Environmental aspects, technological achievements and challenges. Renew. Sustain. Energy Rev. 75, 35-45. 11. Galland-Irmouli, A.V., Fleurence, J., Lamghari, R., Lucon, M., Rouxel, C., Barbaroux, O., Bronowicki, J.P., Villaume, C., Gueant, J.L., 1999. Nutritional value of proteins from edible seaweed Palmaria palmata (Dulse). J. Nutr. Biochem. 10, 353–359. 12. Gardiner, G.E., Campbell, A.J., Doherty, J.V.O., Ross, R.P., Lawlor, P.G., 2008. Effect of Ascophyllum nodosum extract on growth performance , digestibility , carcass characteristics and selected intestinal microflora populations of grower – finisher pigs. Anim. Feed Sci. Tech. 141, 259–273. 13. Habibi, Y., Lucia, L.A., Rojas, O.J., 2010. Cellulose nanocrystals: Chemistry, self-assembly, and applications. Chem. Rev. 110, 3479–3500.
18
14. Holdt, S.L., Kraan, S., 2011. Bioactive compounds in seaweed: Functional food applications and legislation. J. Appl. Phycol. 23, 543–597. 15. Ivanova, V., Rouseva, R., Kolarova, M., Serkedjieva, J., Rachev, R., Manolova, N., 1994. Isolation of a polysaccharide with antiviral effect from Ulva lactuca. Prep. Biochem. 24, 83– 97. 16. Jang, J.S., Cho, Y.K., Jeong, G.T., Kim, S.K., 2012. Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica. Bioprocess Biosyst. Eng. 35, 11–18. 17. Jaulneau, V., Lafitte, C., Jacquet, C., Fournier, S., Salamagne, S., Briand, X., EsquerréTugayé, M.T., Dumas, B. 2010. Ulvan, a sulfated polysaccharide from green algae, activates plant immunity through the jasmonic acid signaling pathway. J. Biomed. Biotechnol. 2010, 111 18. Kaeffer, B., Bénard, C., Lahaye, M., Blottière, H.M., Cherbut, C. 1999. Biological Properties of Ulvan, a New Source of Green Seaweed Sulfated Polysaccharides, on Cultured Normal and Cancerous Colonic Epithelial Cells. Planta Med. 65, 527–531. 19. Konda, N.V.S.N.M., Singh, S., Simmons, B.A., Klein-Marcuschamer, D., 2015. An Investigation on the Economic Feasibility of Macroalgae as a Potential Feedstock for Biorefineries. Bioenergy Res. 8, 1046–1056. 20. Kumari, P., Reddy, C.R.K., Jha, B., 2011. Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae. Anal. Biochem. 415, 134–144. 21. Kwanyuen, P., Burton, J.W., 2010. A Modified Amino Acid Analysis Using PITC Derivatization for Soybeans with Accurate Determination of Cysteine and Half-Cystine TL 87. J. Am. Oil Chem. Soc. 87, 127–132. 22. Lahaye, M., Robic, A., 2007. Structure and function properties of Ulvan, a polysaccharide from green seaweeds. Biomacromolecules 8, 1765–1774.
19
23. Mao, W., Zang, X., Li, Y., Zhang, H., 2006. Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity. J. Appl. Phycol. 18, 9–14. 24. Mata, L., Magnusson, M., Paul, N.A., de Nys, R., 2016. The intensive land-based production of the green seaweeds Derbesia tenuissima and Ulva ohnoi: biomass and bioproducts. J. Appl. Phycol. 28, 365–375. 25. McPherson, R., 1993. Hand book of dietary fiber in human nutrition, in: G.A.S., S. (Ed.), CRC press, Boca Raton, FL, pp. 7–11. 26. Mihranyan, A., Llagostera, A.P., Karmhag, R., Strømme, M., Ek, R., 2004. Moisture sorption by cellulose powders of varying crystallinity. Int. J. Pharm. 269, 433–442. 27. Mišurcová, L., Ambrožová, J., Samek, D., 2011. Seaweed lipids as nutraceuticals. Adv. Food Nutr. Res. 64, 339–355. 28. Morelli, A., Chiellini, F., 2010. Ulvan as a new type of biomaterial from renewable resources: Functionalization and hydrogel preparation. Macromol. Chem. Phys. 211, 821– 832. 29. Nelson, M.M., Phleger, C.F., Nichols, P.D., 2002. Seasonal lipid composition in macroalgae of the northeastern Pacific Ocean. Bot. Mar. 45, 58–65. 30. Oksman, K., Aitomäki, Y., Mathew, A.P., Siqueira, G., Zhou, Q., Butylina, S., Tanpichai, S., Zhou, X., Hooshmand, S., 2016. Review of the recent developments in cellulose nanocomposite processing. Compos. Part A Appl. Sci. Manuf. 83, 2–18. 31. Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E., Osorio, A., Rios, A., 2006. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chem. 99, 98–104. 32. Papadopoulos, G., Goulas, C., Apostolaki, E., Abril, R., 2002. Effects of dietary supplements of algae, containing polyunsaturated fatty acids, on milk yield and the composition of milk products in dairy ewes. J. Dairy Res. 69, 357–365.
20
33. Parikh, A., Madamwar, D., 2006. Partial characterization of extracellular polysaccharides from cyanobacteria. Bioresour. Technol. 97, 1822–1827. 34. Pengzhan, Y., Ning, L., Xiguang, L., Gefei, Z., Quanbin, Z., Pengcheng, L., 2003a. Antihyperlipidemic effects of different molecular weight sulfated polysaccharides from Ulva pertusa (Chlorophyta). Pharmacol. Res. 48, 543–549. 35. Pengzhan, Y., Quanbin, Z., Ning, L., Zuhong, X., Yanmei, W., Zhi'en, L., 2003b. Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity. J. Appl. Phycol. 15, 21–27. 36. Prasad, K., Das, A.K., Oza, M.D., Brahmbhatt, H., Siddhanta, A.K., Meena, R., Eswaran, K., Rajyaguru, M.R., Ghosh, P.K., 2010. Detection and quantification of some plant growth regulators in a seaweed-based foliar spray employing a mass spectrometric technique sans chromatographic separation. J. Agric. Food Chem. 58, 4594–4601. 37. Robic, A., Sassi, J.F., Dion, P., Lerat, Y., Lahaye, M., 2009. Seasonal variability of physicochemical and rheological properties of ulvan in two ulva species (chlorophyta) from the Brittany coast1. J. Phycol. 45, 962–973. 38. Simopoulos, A.P., 2002. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed. Pharmacother. 56, 365-379. 39. Santoso, J., Gunji, S., Yoshie-Stark, Y., Suzuki, T., 2006. Mineral Contents of Indonesian Seaweeds and Mineral Solubility Affected by Basic Cooking. Food Sci. Technol. Res. 12, 59–66. 40. Sharma, L., Banerjee, M., Malik, G.C., Vijay Anand, K. Sudhakar, G., Zodape T., Ghosh A., 2017. Sustainable agro-technology for enhancement of rice production in the red and lateritic soils using seaweed based biostimulants. J Clean Prod. 149, 968-975
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41. Singh, A.P., Avramis, C., Kramer, J.K.G., Marangoni, A.G., 2003. Algal meal supplementation of the cows’ diet alters the physical properties of milk fat. J. Dairy Res. 71, 66–73. 42. Singh, S., Singh M.K., Pal, S.K., Trivedi, K., Yesuraj, D., Singh, C.S., Vijay Anand, K.G, chandramohan, M., Patidar, R., Kubavat, D., Zodape, S.T., Ghosh, A., 2016. Sustainable enhancement in yield and quality of rain-fed maize through Gracilaria edulis and Kappaphycus alvarezii seaweed sap. J Appl Phycol. 28, 2099-2112. 43. Trivedi, N., Baghel, R.S., Bothwell, J., Gupta, V., Reddy, C.R.K., Lali, A.M., Jha, B., 2016. An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass. Sci. Rep. 6, 30728. 44. Wang, D., Wang, L. jun, Zhu, F. xue, Zhu, J. ye, Chen, X.D., Zou, L., Saito, M., Li, L. te, 2008. In vitro and in vivo studies on the antioxidant activities of the aqueous extracts of Douchi (a traditional Chinese salt-fermented soybean food). Food Chem. 107, 1421–1428. 45. WHO. 2003. Diet, nutrition and the prevention of chronic diseases. WHO technical report series 916. Geneva: WHO. 46. Yaich, H., Garna, H., Bchir, B., Besbes, S., Paquot, M., Richel, A., Blecker, C., Attia, H., 2015. Chemical composition and functional properties of dietary fibre extracted by Englyst and Prosky methods from the alga Ulva lactuca collected in Tunisia. Algal Res. 9, 65–73. Figures captions: Figure 1 Data on protein content at different biorefinery stages Figure 2 Data on protein recovery efficiency Vs biomass consumption at different stages of biorefinery
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Highlights • • •
Biorefinery approach for integrated extraction of proteins from marine macroalgae The digestibility of protein is more than 85% based on in vitro enzyme hydrolysis Sap extraction was optimized for effective recovery of constituents from biomass
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