Finding valuable bioactive components from Jerusalem artichoke (Helianthus tuberosus L.) leaf protein concentrate in a green biorefinery concept

Jerusalem artichoke is widely known for its inulin-enriched tubers. Recently the opportunity has been arisen to involve the whole plant in biorefinery concept due to its high lignocellulose biomass and tuber production. This paper focuses on the repeatedly harvestable green biomass of Jerusalem artichoke. Ultra-High Performance Liquid Chromatography-Electrospray Ionization/Mass Spectrometry (UHPLC-ESI-MS) was applied to identify the phytochemicals in Jerusalem artichoke leaf protein concentrate (JAPC) thermally extracted from green biomass of three clones, i.e., Alba, Fuseau and Kalevala. Amino acid and fatty acid profiles as well as yield of JAPC were also analyzed. The UHPLC-ESI-MS analyses showed that no toxic phytochemicals were identified in JAPC. The results revealed, also, that JAPC is not only essential-amino acids-rich but also contains substantial amounts of polyunsaturated fatty acids (66-68%) such as linolenic and linoleic acids. Linolenic acid represented 39-43% of total lipid content; moreover, the ratio between ω-6 and ω-3 essential fatty acids in JAPC was ∼0.6: 1. Using UHPLC-ESI-MS, the following hydroxylated methoxyflavones were for the first time identified in JAPC, i.e., dimethoxy-tetrahydroxyflavone, dihydroxy-methoxyflavone, hymenoxin and nevadensin. These compounds are medically important since they are referred to as anti-cancer, anti-inflammatory and antioxidants. Also, liquiritigenin - estrogenic-like compound - was identified in JAPC alongside the following terpenes, i,e., loliolide and dihydroactinidiolide. However, no remarkable differences of phytochemicals, fatty acids and amino acids composition were seen among Jerusalem artichoke clones. The green biomass of tested clones ranged between 5 to 5.6 kg m-2 and JAPC yield varied from 28.6 to 31.2 g DM kg-1 green biomass with total protein content, on average, of 33.3%. According to our knowledge, this paper is the first scientific report highlighting bioactive substances in JAPC such as PUFA phytochemicals. These results clearly prove that JAPC is a valuable product which can direct towards human and animal nutrition as well as it can serve as basic material for different industrial purposes.


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Ensuring an adequate supply of protein has become one of the challenges facing the world today, 48 which is expected to worsen in the future as a result of the terrible increase in population and the 49 erosion of agricultural land. The global protein supply relies on vegetal sources (57%), meat 50 (18%), dairy products (10%), fish and shellfish (6%) and other animal products (9%), 51 respectively [1]. Depending on species different organs of plants can serve as protein source such 3 52 as seed (e.g. soy; pea; almond; pea; rice; wheat as seed-based protein), fruit (e.g. cranberry fruit-53 based protein), leaf (e.g. moringa leaf-based protein) and root (e.g. maca root-based protein) 54 [2,3]. Recently, leaf-based protein has gained an intensive attention. Alfalfa and grasses are the 55 most perspective species in continental climate zone. Alfalfa protein-xanthophyll concentrate 56 (APC) is an extensively studied processed product of fractionated green biomass. It has already 57 manufactured in different countries such as France for feed and food purposes [4,5]. 58 In context to leaf-based protein, the concept of green biorefineries is not be bypassed. Green 59 biorefineries are novel technology systems for production of materials and energy processing 60 using parts or total green plants [6]. Above all, green biorefinery technologies are based on 61 traditional technologies of green forage preservation, leaf-protein extraction, chlorophyll 62 production, and modern biotechnological and chemical conversion methods [6,7,8,9]. Sugar beet, 63 clover, alfalfa and grass are the most common and perspective species for green biorefinery 64 purposes in continental climate zone. However, several other crops may also be suitable. 4 75 has widely gained the attraction of many researchers for bioethanol production due to its 76 lignocellulose content [12,17], it can be directed towards other significant uses such as animal 77 feeding as fresh forage, silage, or food pellets [16]. In addition, there are some information about 78 green leafy shoot which contain protein (stalk: 1.6%-4.5% DW; leaves 7.1%-24.5% DW), 79 volatile sesquiterpenes, some phenolic compounds, chlorophylls, carotenoids [11,17,18]. 80 However, direct consumption of fresh or dried Jerusalem artichoke biomass is not preferable 81 because trichomes covered leaves and stem [11]. Alternatively, green biomass can be fractionated 82 mechanically to green juice and fiber fractions. The cell wall-deprived green juice can be 83 thermally treated in order to extract proteins by coagulation. From the aspect of green biorefinery 84 the separate leaf protein concentrate as main product has special importance hence to become 85 competitive process it should produce at least one product of high value (such as a high value 86 chemical or material). 87 In accordance with the above, the present study aimed to provide a detailed insight into the  clones started on 5 th April 2016 using size-identical tubers (60 -80 g/tuber). No irrigation and 106 fertilization were applied in the plantation during the growing season. Chemical characteristics of 107 the experimental soil were: total N (555±2 mg kg -1 ); total P (6793±17 mg kg -1 ); total K (1298±7 108 mg kg -1 ) and humus (1.9±0.02%). 110 Considering the re-growing ability of Jerusalem artichoke plants, the green biomass of the three 111 clones was harvested two times during the growing season when young shoot reached 1.3 -1.5 m 112 height from soil surface. The first harvest was carried out on 27 th June 2016, while the second 113 harvest was on 8 th August 2016. Fresh yield of aerial part was measured.

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Fractionation of harvested green biomass 115 The harvest of Jerusalem artichoke plants was carried out in the early morning and immediately 116 transferred to the laboratory in an ice box to inhibit the degradation chemical compounds. Plants 117 were harvested 20 cm above soil surface. One-kilogram green biomass was mechanically 6 118 pressed and pulped by a twin-screw juicer (Green Star GS 3000, Anaheim, Canada) to green juice 119 and fiber fractions in three replicates. Thereafter, the green juice was thermally coagulated at 80 120°C to obtain JAPC. The JAPC was separated from brown-colored liquid fractions using cloth 121 filtration. Both fresh and dry masses of JAPC were weighted. The JAPC was lyophilized using 122 the Alpha 1-4 LSC Christ lyophilizer.  set to 30 NCE. The difference between measured and calculated molecular ion 186 masses was less than 5 ppm in every case. The data were acquired and processed using Thermo    Results of aboveground fresh biomass yield presented in Table 1. It is showed that the yield of 207 Jerusalem artichoke clones were similar. No significant differences among tested clones (i.e., 208 Alba, Fuseau and Kalevala) were noticed especially during the 1 st harvest. By contrast, the  Table 1). The calculated total aboveground fresh biomass yield -as an average -was estimated 213 to be 7.7 kg m -2 . Table 1. Aboveground fresh biomass, dry mass and total protein content of Jerusalem artichoke  Table 1. Statistical analysis showed insignificant differences among 220 Jerusalem artichoke clones whether in 1 st or 2 nd harvests. The JAPC yield ranged from 28.3 221 (Fuseau) to 32.3 (Kalevala) g kg -1 fresh biomass in the 1 st harvest, while in the 2 nd harvest JAPC 222 yield varied from 28 (Kalevala) to 30.4 (Alba) g kg -1 fresh biomass (Table 1)  The total protein content (m/m%) of JAPC generated from fresh green biomass of Jerusalem 228 artichoke clones ranged between 33.3 m/m% (Fuseau) and 35.3 m/m% (Alba) in the 1 st harvest, 229 while in the 2 nd harvest it varied from 31.6 m/m% (Alba) to 35.2 m/m% (Fuseau). Statistically, 230 no significant differences were calculated either between clones or between harvests (Table 1).

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The average total protein content in the 1 st harvest was 34.1 m/m% and in the 2 nd harvest was 232 33.4 m/m%. The annual average of total protein content of JAPC extracted from Jerusalem 233 artichoke fresh biomass is estimated to be 33.8 m/m% (Table 1).

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The results of amino acid composition of JAPC obtained from green biomass of Jerusalem 236 artichoke clones are presented in (Table 2). The essential amino acids (i.e., lysine, histidine, 237 isoleucine, leucine, phenylalanine, methionine, threonine and valine) play a major role in feeding;  (Table 2). The concentration of lysine as essential amino acid has special importance in 243 animal feeding. We found that the lysine content of Alba, Fuseau and Kalevala (ranged between  (Table   248 2). Results, also, showed that lysine content increased in the 2 nd harvest compared to 1 st harvest 12 249 for all clones; while, a reduction in methionine content was found in the 2 nd harvest compared to

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Among monosaturated fatty acid (MUFA) the oleic acid (C18:1ω-9) could be detected with 264 higher values (6.6 -11.6%). The proportion of palmioletic acid (C16:1ω-7) was significant 265 lower, changed in the range of 0.7 -1.1% (Fig 3).   (Table 3). Table 3 showed that the phenolic compounds were significant part of the     When it comes to the ideal protein source, the amino acid profile cannot be ignored because 385 among the 20 proteinogenic amino acids nine cannot be synthetized by most animal species [31].

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The ratio of these essential amino acids has special interest. Among green biomass originated 387 fractions, JAPC as dedicated protein enriched product for feed was examined more thoroughly.    (Table 3) parameters, were similar in JAPC generated from Jerusalem artichoke clones. Present 517 biochemical analysis revealed that the JAPC is not only a good source of protein with favorable 518 amino acid composition but also it is repository of essential fatty acids, flavonoid and non-519 flavonoid phytonutrients. We found that the quantity and/or quality of phytochemicals are 520 specific primarily for the Jerusalem artichoke species and for the technological way. Within the 521 species, slight difference can be revealed in the examined parameters between the clones. Results

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of present work confirm that this underestimated plant can be directed not only towards tuber 523 production for inulin extraction, but the green biomass can also represent a value for JAPC 524 production under low inputs in green biorefineries.