Owen Mccafferty'

Mojo Mickybo

Ex-centric Didactic Drama

Keywords: didacticism, neo-Brechtian drama, Northern Ireland, sectarianism, typography

Protestant Mojo remembers his teens in 1970 Belfast when he made friends with Catholic Mickybo. Yet their friendship was brought to an end after the murder of Mickybo's father by UDA men, which shows that sectarianism does not spare children. This play borrows many techniques from Brechtian epic drama that the playwright transposes to a new context. In the written version of the play, this neo-Brechtian use of dramatic devices is also conveyed through the absence of some typographical elements which particularly stands out in the dialogues. McCafferty obviously rejects the typographical norm that should be abided by when writing. We consider this overt ex-centricity on McCafferty's part as an aesthetic act of resistance to denounce the Troubles, violence and its cycle of repetition. The playwright aims to use neo-Brechtian techniques so as to underline the post-colonial dimension of his play and the need to get away from sectarianism. In this respect, the play becomes didactic.

spaces between and around them); secondly, the writing rules, the aspect of the signs assembled in words, sentences, paragraphs. Punctuation naturally belongs to this typographical code: it is part of the language structure in so far as it introduces the articulations, the breaks, the breaths, within a text. With typography, the text is given a voice and the message delivered can be harder-hitting, fiercer, or softer. The tone of this metalanguage is further given by the font. The choice of Belfast playwright Owen McCafferty not to stick to all the rules of this particular code in Mojo Mickybo shows his desire to resist what is imposed to him: bad typography resists good typography. This also means that through his style, McCafferty subtly aims at showing his resistance to sectarianism in Northern Ireland. Our focus will thus be on the absence of some typographical rules, which mainly have to do with punctuation and capitalization, and their meanings in the context of "performative writing" (Worthen 2005, 14). This, added to other devices borrowed from Brechtian theory, takes a neo-Brechtian dimension and reinforces the idea that Mojo Mickybo exemplifies Brecht's epic drama. The playwright's overt ex-centricity is an aesthetic act of resistance to denounce the Troubles, violence and its cycle of repetition. McCafferty uses neo-Brechtian techniques so as to point out the post-colonial dimension of his play and the need to get away from sectarianism.

In this article, we shall study the neo-Brechtian redefinition of Northern Irish drama through McCafferty's ex-centricity in Mojo Mickybo's text and performance. This study sheds light to the reasons why the missing typographical elements add to the realistic representation of the Troubles on stage and illustrate Brecht's epic theory. The play's neo-Brechtian quality, conveyed through fragmentation, juxtaposition, alienation and historicisation in particular, indeed strengthens its post-colonial message. This present analysis will demonstrate that examining both the performance and the play text from a neo-Brechtian perspective allows for their complementary nature, the neo-Brechtian apparatus being possibly used to explore both a script and a performance. Henceforth, McCafferty's goal is not only overtly aesthetic; it is also covertly political and didactic.

Fragmenting the world

Brecht's theory about epic drama was based on the idea that the world was fragmented. Reality should be represented as such in drama; as if it were possible to "take a pair of scissors and cut it into individual pieces which remain fully capable of life." (Brecht, 1964: 70). In McCafferty's play, fragmentation, which is found in the contents since it deals with topics such as the religious divide and the Troubles inextricably linked to that religious divide, is also exemplified by the form.

On the first pages preceding the play text, McCafferty warns the readers that Mojo Mickybo is a "play for two actors" who "should divide the characters" (8). Both actors should be in their late thirties/ early forties, but they also play the roles of ten year-olds and elderly persons. The actor embodying Mojo is also the narrator, and thus narrates the story as he lived through it. Brecht's theory put forward the idea that Man should be presented as fragmented. The German playwright noted that "the continuity of the ego is a myth. A man is an atom that perpetually breaks up and forms anew." (Brecht 1964, 15). Similarly, post-colonial theories consider the body of the actor as a site for "resistant inscription" since it "disrupts the constrained space and signification left to it by the colonizers" (Gilbert and Tompkins 2006, 204). Deciding that one actor will play the role of many characters including a narrator, McCafferty demonstrates the possible sites of resistance and shows the fragmentation within a character, the "multiple entities that constitute a social subject" (Gilbert and Tompkins 2006, 232). Hence the combination of these devices hindering the unitary view that one could have on a character.

Besides, the narrative technique chosen by the playwright to tackle the subject of childhood during the Troubles is also one of the devices creating fragmentation within the play. At its very beginning, the two characters, Mojo and Mickybo represented in their teens, open the play. Their dialogue is immediately followed by the comment of the narrator, who is in fact Mojo once he is an adult: MOJO. mojo MICKYBO. mickybo MOJO. mickybo mojo MICKYBO. mojo mickybo Mickybo is heading a football against the wall. NARRATOR. belfast -the summer of 1970 -the heat's meltin the tarmac on the street the buses are burnin bright an punters are drinkin petrol outta milk bottles -this is where mojo an mickybo used to play (9). The narrator's comments on the past of both children, his numerous flashbacks and flash forwards give time a fragmented aspect.

Similarly, space is submitted to fragmentation. The place of the narrator (which could be anywhere in Belfast) and the space of his story (the streets of east Belfast) are not the same, and the places within the story of the narrator, back in the 1970s, also differ. Sometimes readers are warned in the stage directions of their changes, but most of the time they are not told anything and it is up to the actors and their performance on the stage to show them when the play is staged.

If the fragmented contents of this piece is meant to echo the fragmented situation of Belfast during the Troubles, then it is also conveyed through its structure. The dialogues are often interrupted by either the intervention of the narrator or songs that can be sung by all the characters, including the narrator, like on this occasion:

NARRATOR. mojo galloped back up the road thinkin mickybo was a geg (Sings.) rain drops keep fallin on my head -because i'm free nothin worrin me [...] (20).

As a matter of fact, songs were part of a series of tools advocated by Brecht in epic drama. In Mojo Mickybo, they are always referred to as sections which are sung. We know when they start, which is written in the stage directions. Yet nothing is said about their ending as we can see in this excerpt when Mojo remembers Mickybo's mother: MICKYBO'S MA. would you like to hear my plan son -i was sittin on top of a mountain of dishes the other night listening to elvis on the radio and thinking of the time when the man that i love header and all that he is used to take me dancing -(Sings.) oh how we danced on the night we were wed we danced and we danced cause the room had no bed -there was this strange noise come out of the radio it sounded like the king had eaten something very large that didn't agree with him and was choking on his own own boke -then a voice said we come in peace earth people if you lose your head you lose your money -things may be getting a bit hairy but we're here to save you all especially wee mickybo […] (31).

In Brechtian theory, "songs", in addition to being an aesthetic innovation which fragments the play, are meant to criticize the external contemporary world. So when Mickybo's mother mentions the words "peace", "money" and uses the verb "save", we cannot but understand the poor economic situation of the Catholics (and Protestants alike) beset by violence at that period of the Troubles.

Since the "mise-en-page [is also] a site of performance" (Worthen 2005, 11), any playwright should aim at getting the contents and the form to coincide. In Mojo Mickybo, fragmentation equally pervades the form visually. The reader of the play cannot but be struck by the numerous dashes which do not give the text a flowing, homogeneous aspect like in the following lines: MICKYBO. Everybody knows rip the balls -he puts black boot polish in his hair an doesn't wear no socks -an my da says he pisses in the sink cause he couldn't be fucked to go out to the yard -nobody's ever saw im but me -I saw im buryin dead rats over the timbers -wanna dig with the stick? (12).

These dashes are here meant to reflect the unstable situation of Northern Ireland as experienced by the two communities all the more so as each clause separated by a dash puts to the fore the idea that there is not only one perspective, there are several viewpoints. In the case of the above example -and this is a recurring device -we get the standpoint of Mickybo, his father, and eventually the narrator since he filters the whole stories retrospectively. For Brecht, reality did not have any centre of action but many, independent the ones of the others, yet they made up a unity in the end. Throughout this play, this idea is highlighted with these numerous markers, separating the sentences and playing their roles of inserting precisions within a sentence as well as contrasting values, opposing opinions.

The dashes, replacing all other signs of punctuation, give the text a fragmented aspect and break the dramatic illusion of reality as first put forward by Brecht. Not only are they meant to reproduce reality on the stage but they also entice the audience to realize that this reality is precisely performed. This is here an attempt on the author's part to redefine Northern Irish drama about the Troubles. Having the play performed for five years in a row (from 1998 to 2003) by the Northern Irish theatre company Kabosh, renowned for pushing the limits of performance in an innovative visual and physical way, enhances this experimental quality. Fragmentation is indeed part of a broader project which Brecht longed for. Throughout his work the German playwright wanted to "show things as they are" (Brecht 1964, 15) but not in a mimetic way. He introduced distance so that spectators had a better and impartial vision of reality. This is also what McCafferty requires from his audiences.

Taking some distance

To create distance, Brecht used the techniques of juxtaposition and montage in his plays, so does McCafferty. The juxtaposition of sentences is particularly relevant in the dialogues of Mojo Mickybo where parataxis is omnipresent. There is no precise outlining of syntactic patterns, particularly in embedded free indirect speech. This is the case in the quote mentioned above by Mickybo's mother when she was reported to have said: "then a voice said we come in peace earth people if you lose your head you lose your money" (31). The juxtaposition of the grammatical subjects and more particularly of the pronouns "we" and "you" hampers the fluidity of the sentence, which arouses the attention of the public.

McCafferty also juxtaposes different layers of reality. We first know the narrator's situation (1998 Belfast) then that of the children (1970 Belfast). Within this particular reality, we get the viewpoints of different people all embodied by the two actors on stage. These worlds are different from that of the spectators (no matter the time, no matter the place of the performance) and that of the readers (since reading it could occur at any time in any place).

Juxtaposition is also present in the structure of the play since the public is never warned of the changes of time and place. Speech switches from the children and the various characters to the narrator and it is up to the performers to indicate it through their performance:

MICKYBO. you shit yourself MOJO. you do MICKYBO. you do MOJO. kack the breeks MICKYBO. shit the trunks NARRATOR. mojo mickybo -thick as two small thieves-the greatest lads god ever pumped breath into -the day they met was the hottest ever in the whole christendom [...] the world draggin itself along like it was out of breatha belter MOJO. many ya done now mickybo? MICKYBO. three hundred an twenty-four -twenty-five -twenty-six... (10).

The climax of this juxtaposition of space and time emerges when Mojo narrator asks a question to which Mickybo as a child answers: NARRATOR (sings.). don't ever hit your granny with a shovel, it leaves a dull impression on her mind -what happens mickybo when ya hit your granny with a shovel? MICKYBO. her eyes pop out an her face goes like that (Grimace.) (17) This example, linking the time and space of the narrator (1998) to those of the children (1970), proportionally sheds light on the violent environment that surrounds the children. Yet, this conversation is not real and could never have happened since the public is told that Mojo and Mickybo did not see each other anymore in the 1990s: NARRATOR. love many trust few and learn to paddle your own canoe -years later i was walking through the town -this town -belfast -a town with memories -i saw mickybo across the street -mojo mickybo MICKYBO. mickybo mojo NARRATOR. we both pretended we didn' t know each other and walked on -mojo mickybo (49). This device on McCafferty's part further breaks the illusion of realism and affects both the actor and the spectator as the analysis of the articulation of the play text and the performance confirms.

Worthen writes that "attending to the material form of plays in print may also provide a means to 'alienate' and so to observe, other aspects of our understanding of dramatic performance, the interplay between the text and the naturalized strategies of its production onstage." (Worthen 2005, 61-62). One of the processes creating distanciation is alienation, a key-notion of Brechtian theory according to Elin Diamond. In an article entitled "Brechtian theory/ Feminist theory", she explains that "the cornerstone of Brecht's theory is the Verfremdungseffekt, the technique of defamiliarizing a word, an idea, a gesture so as to enable the spectator to see or hear it afresh." (Diamond 1988, 84). Thanks to Brechtian distanciation, the spectator and the reader become aware of the nature of this relation, of this alienation, this alienating environment and "allow [them] to criticize [it] constructively from a social point of view" (Brecht 1964, 125). In fact, with this piece, McCafferty offers the public a new perspective on the Troubles through the friendship of two working-class children, whose relationship is solidified by a movie, Butch Cassidy and the Sundance Kid. This film, which they keep on going and watching, puts the stress on their desire but impossibility to escape their environment. It enables them to get away from the real world, but only for a short time, as the narrator observes: NARRATOR. half time -back to the real world -decisions have to be made -important decisions that would give a book a headache -who's who an what's what (17).

Readers do not know if the movie can be broadcast on the stage; yet, if it is possible in some performances, then this montage creates even more distance and alienation.

The A-effect in the performance does not only concern the distance set up between the spectators and the play; it is also found in the relationship between the actors and their own roles. From the beginning, we are told that the two actors embody all the characters, be they adults or children. For practical reasons, the actor incarnating Mojo plays the role of Mickybo's parents, and that embodying Mickybo plays the roles of Mojo's parents. Therefore the actors cannot possibly identify with their characters since it would be difficult to share the viewpoints of Protestants and Catholics at the same time. This distance the performers must dramatize is also echoed by the numerous dashes that fragment the play. They materialize the possible slippages from one performer to one character, from one character to another performer and thus participate to creating a neo-Brechian A-effect.

The A-effect is thus further found in the written version of a play and specifically suggested by typography. If Worthen observes that "the materiality of the poem on the page alienates language from its typical, commodified usage" (Worthen 2005, 135), it is first the absence of capitals in Mojo Mickybo that catches the reader's attention. in A Comprehensive Grammar of the English Language, Randolph Quirk et al. remind us of the use of capitals as follows: "in addition to marking the beginning of a sentence, initial capitals are used for specifying proper nouns for example, persons, places, works of literature, days of the week, months of the year […]" [START_REF] Randolph | A Comprehensive Grammar of the English Language[END_REF](Quirk et al. , 1638). Yet, in the dialogues, McCafferty omits the capital letters on the names of people. Readers can immediately detect it when the two main characters introduce each other at the very beginning of the play: MOJO. mojo MICKYBO. mickybo MOJO. mickybo mojo MICKYBO. mojo mickybo (9).

Right from the start the narrator aims to consider the two boys as one entity and juxtaposes their Christian names. Likewise, geographical names, such as "belfast" (9) or "australia" (12), do not have any capital letters. They are all treated as if they were common names. Furthermore, there is no capital letter on the religious denominations "catholic" and "protestant" when there should be. Through this absence of capitalization, the author seems to disclose his desire to forget the notion of hierarchy, the difference between the two religious communities that faced each other in the 1970s in Northern Ireland. On the contrary, he seems to be lauding equality. From a Brechtian perspective, these missing capital letters give the text a strange aspect. It is as if the text was alienated along the Brechtian definition of alienation, as "allow[ing] us to recognize [a] subject, but at the same time mak[ing] it unfamiliar." (Brecht quoted in Diamond 1988, 84). In fact, readers are not used to reading a text completely devoid of capital letters.

McCafferty confides that one of his objectives was to have all his characters speak in a "heightened Belfast dialect" so as to "try to create a new Belfast theatrical speech"(<http://www.culturenorthernireland.org/article.aspx?art_id=1894>) but he had to set aside a lot of linguistic rules to reach his aim. His choice is an act of resistance to specific imposed and institutionalized codes like grammar, punctuation, syntax. That is why, McCafferty's public can read and hear sentences such as "that's borin -yer da's borin mojomon we'll go over the timbers an burn wood" (30) which convey a realistic impression of spoken vernacular. According to Gilbert and Tompkins, "post-colonial stages are particularly resonant spaces from which to articulate linguistic resistant to imperialism" (Gilbert and Tompkins 2006, 166). Therefore, the dialect spoken by the two children acts as a political medium with a meaning in itself. It is strengthened by the lack of punctuation to orient the performance. Worthen calls this style the "performative print style" and gives the example of George Bernard Shaw: A play's language does not only live in the mind's eye, it also lives in the ear and on the tongue. Shaw's consistently rhetorical use of punctuation -using punctuation to mark the rhythms of speech rather than the logic of syntaxand his celebrated use of dialect might well be read as traces of the stage, or as places where Shaw uses the accessories of the page actually to direct the performance. The tension between rhetorical and syntactic pointing is one of the places where the text's representation of the dramatic fiction joins its implication of performance, its way of specifying action on stage. (Worthen 2005, 56).

On the one hand, for Worthen, "language writing alienates language because it is an alternative language system" (Worthen 2005, 126). On the other hand, Brecht saw language as possibly "alienated by translation into the actor's native dialect" (Brecht 1964, 139). Confronted to this, the audience become estranged, unfamiliar to McCafferty's alienated rhetoricity; their comprehension of the text is sometimes inhibited and they might even be misled. To give a precise example, "weeker" (13), which sounds like the comparative form of the adjective "wick", meaning "mean" in Northern Irish slang, expresses the exact opposite in the mouth of the two boys. The words of the narrator are thus both alienating and alienated.

The author effectively empowers his narrator. The latter relates all the stories of the two boys and twelve other people without ever quoting them with inverted commas in the written text. This device appears to be close to the epic process of historicisation, another technique to create distance. Indeed, historicisation in Brechtian drama was used so that the playwright might point out to the spectators the place of Man in History, how He transforms the world, how He determines History and how History may determine Him. In this play, the narrator is in charge of articulating public History and private stories, time, place and space to the detriment of the protagonists. This is the reason why there is no capital on the subject pronoun "i" whenever a character speaks, as illustrated in the following quote: MICKYBO. wanna know what i heard? MOJO. wha? MICKYBO. the whole a belfast is goin mad an we're all gonna get murdered in our beds. (30).

In post-colonial theories, histories compete and confront one another and enhance the permeability of space, time and content. The narrator crystallizes all these elements, which creates distanciation in so far as the audience becomes confronted to two or three tenses and places: their own (past, present, future) and that of History, or, as it were, the history of Northern Ireland in the 1970s. When a text is historicised, it is naturally distanced in so far as it shows the various possibilities, which is the case in Mojo Mickybo with the backward glance of Mojo narrator, thirty years after the episodes he is narrating took place. This study lays emphasis on "the semantic value of the accessories of print" (Worthen 2005, 58) since both the text and the performance participate to the good understanding of the message of the author: the post-colonial redefinition of Northern Irish drama thanks to neo-Brechtian devices. However, it is up to the audience to decipher the clues left throughout the play text by McCafferty and meant to be staged. That is why Mojo Mickybo can be seen as a neo-Brechtian Lehrstück, or didactic play.

Teaching audiences

Brecht wanted his theatre to "increase its ability to amuse, and [...] to raise its value as education" (Brecht 1964, 130). These two values, entertaining and instructing, were meant to encourage "the spectator to draw conclusions about how the world works" (Brecht 1964, 150). Like Brecht, McCafferty specifically makes use of dialectics and a direct address to the public to entertain and instruct his audiences.

The German playwright saw in dialectics a means to bring out the truth about social realism. He recommended to confront ideas in an artistic way to lead to debates that should point out the power of Man. Mc-Cafferty, proposing a text alienated, a man (or a child in this case) dominated, changed and divided because of his environment, calls upon his audience's capacity to reflect upon freedom. He starts from his own, giving himself the liberty not to comply with some rules of typography, grammar and syntax in the dialogues, for, effectively, the stage directions, expressing his voice, are not concerned by the absence of any linguistic rule. They indicate that the playwright is still in charge, that he can always control the world he is creating and that he is delivering a message. Through his style, the playwright puts forward the idea of an ideology of freedom in possible reaction against a given political economic and social system. The neo-Brechtian epic style, which Mojo Mickybo illustrates, relies on the double movement of alienation and freedom, of giving up and choosing, of accepting and refusing, which are also encoded in the text through the use of dashes. If the latter reflect fragmentation, they might also give an impression of continuity, of connection between the various characters. Quirk et al. call them "correlative punctuation marks" [START_REF] Randolph | A Comprehensive Grammar of the English Language[END_REF](Quirk et al. , 1629)). They echo McCafferty's goal to build a bridge between the two boys and their environment despite the external tensions; tensions that are also encoded in the text by the author. Worthen effectively writes that "modern drama in print typically frames a dialectical tension between the proprieties of the page and the identities of drama" (Worthen 2005, 62). In McCafferty's piece, tensions can be found between the page and the stage, notably as far as punctuation is concerned: the question arises so as to represent the dashes on the stage. If the actors might embody the tensions, and so enable them to emerge, the audiences are encouraged to find some appeasement and, most importantly, uncover the coherence of all the stories filtered by the narrator.

For Gilbert and Tompkins, histories in post-colonial drama "compete with each other to form a complex dialectic which is always subject to change as new players enter the fields of representation" (Gilbert and Tompkins 2006, 110). They particularly shed light on the tensions arising from their evolution between past and present and their differences between public and private receptions. Hence the power of the audience. In her book, Bertolt Brecht, Francine Maier-Schaeffer notes that the epic form of drama gives us the formal means likely to re-centre observable facts which are not natural phenomena and can eventually be changed by Man. Brecht's conception of Man and of the world was philosophical; for him, Man had to be changing. He noted that "changes in his exterior continually lead to an inner reshuffling" (Brecht 1964, 15). So the world had to be completed and the role of drama was to show this possible transformation. The dialectical method was the a tool that Brecht could think about when it came to grabbing reality so as to change it thereafter.

McCafferty shares the German playwright's opinion. He sheds light on the changing nature of a child, in this case, Mickybo, who first went beyond the divide and then got closer to children from his "tribe" -Gank and Fuckface, his previous enemies -after his father died. The narrator explains: NARRATOR. mojo mickybo -great lads -mickybo's in the hut along with gank and fuckface -they're smoking fegs an talking the talk of men -it's showtime (48).

Mickybo even accuses Mojo of stealing his bike. Nevertheless, Mickybo could have resisted it. In his play, McCafferty suggests that one's future has alternatives. Indeed, if we adopt a Brechtian perspective, McCafferty here presents the audience with past facts and their outcome, so that, after taking them into account, they may learn that the future could have been different, and that the cycle of endless violence could have been broken back then. The personal futures of the children could have changed had Mickybo acted differently at his father's death, or even later. As a matter of fact, between then (the time when the children met) and now (the time it is narrated), there have been many changes in Northern Ireland. The peace process had already started. Yet, Mojo has still been considered as an enemy and has been left on his own. The narrator advises him to: "love many trust few and learn to paddle your own canoe" (49). Mickybo's decision could also have influenced the future of their communities if we consider Mojo and Mickybo to be allegorical characters speaking for their respective community. For Brecht, epic drama triggers off the morale of History, but does not speak for History, rather for the victims. In McCafferty's play, both Mojo and Mickybo, representing their communities, can be held as victims of History and place.

Gilbert and Tompkins explain that "post-colonial spatial histories dramatise the dialectic of place and displacement" (Gilbert and Tompkins 2006, 156). In the play under scrutiny here, the movie the two children are keen on watching becomes a parable of their relationship to their environment. The lives of the cow boys appeal to the children as the narrator says:

NARRATOR. butch cassidy an the sundance kid -a fine feelin it must be to be a cowboy -money in yer pocket a horse on yer arse an a gun in yer holster -but times are hard -there's no ham for the sandwiches an torch woman is in the box office paying her dues (16).

Similarly, the countries that attract them most, Bolivia and Australia, represent places where life seems to be better. Francine Maier-Schaeffer explains that parables are at the heart of the dialectical relationship between the general and the particular -which is a basic principle in Brechtian theory. In McCafferty's play, the parable forges a relationship between the Northern Irish communities (the general) and the two boys (the particular).

If the audience is never warned of the shift from one situation to another, from one character to another, from one episode to another, because of the little information they are given and the lack of any institutionalized norm that could orient their thoughts, it is up to them to follow the play with careful attention and bridge all the gaps. This process on McCafferty's part, as well as the intervention of a narrator commenting upon the story of two children years later, echo the abolition of the fourth wall that Brecht advocated and which is another image for getting people closer in a post-colonial context. As a matter of fact, in focusing their attention on what is said by whom, the readers and the spectators are fully implicated in the action and are asked to react. The narrator helps the audience understand their participation. He directly asks them : "know what a mean"(10). For Brecht, the audience must learn by themselves. This is what the German playwright called the play's didactic quality. In a book entitled Lectures de Brecht, Bernard Dort specifies the idea that the strongest ideological message is delivered through didactic plays which are indeed the most perfect realizations of epic plays. When didactic plays are performed, the gap between the playwright and its readers, the actors and the spectators of drama, the actors and the spectators of life, between drama, fiction and reality, between philosophy and politics is filled. Positioned against capitalism, Brecht's aim was to prevent drama lovers from being mere consumers. Similarly, McCafferty invites his audience to meditate upon humanity through this play. Readers and spectators must understand and learn some information by themselves, for, it goes without saying that teaching is closely linked to learning.

When the narrator addresses the public directly, he entices them to question themselves. As a matter of fact, the interrogation mark is the other punctuation mark that we come across in Mojo Mickybo's dialogues. It is also used by the author to enhance the innocence of the two boys, a virtue to which McCafferty pays careful attention. In a private interview, McCafferty confided that it was crucial for him to see the Troubles through the prism of childhood since there is an articulation between segregation and the innocence of the children. He explains: "The reason the play is seen through the eyes of children is that I wanted to show the absurdity of sectarianism. And I thought the best way to do that was through the innocence of children." Both children are indeed eager to have all their questions answered. Some questions even startle the audience because they have nothing to do with the main action. For instance, when Mojo keeps on asking "are wasps bees ?" (37), the audience is expected to react. And so are the actors. Indeed, Maier-Schaeffer explains that didactic plays are revolutionary plays in so far as they also instruct the performers. The play is thus peppered with numerous questions some of which remain unanswered so that audiences and actors might find their own answers.

The study of the absence of typographical rules, notably punctuation, their impact on syntax and grammar, as well as other devices borrowed from Brechtian theory, invites the readers and spectators of the play to understand the message the author delivers. McCafferty's overt ex-centricity, meant to reshape the play aesthetically, strengthens his covert political message: denouncing the conflict. He stages the impossible friendship of two innocent children who managed to go beyond the divide and keep themselves at a distance of the atrocities of the adults' world, that is to say sectarianism and the Troubles. They were caught up by reality but it could have been different. The playwright's ambition is to put forward the mighty power of Man in changing, transforming the world and being transformed. Therefore, the playwright devises new means to reshape the contours of Northern Irish drama, and his use of neo-Brechtian techniques in a post-colonial fashion sheds light on the possible emancipation of Northern Ireland from Great-Britain in the context of the peace process. If having this play printed allows it to be part of literature since "the power of print [is a way] to secure the literary identity of writing" (Worthen 2005, 26), it also guarantees its access across the world over the years. Yet, modifying the rules of print through punctuation and capitalization is a device McCafferty resorted to so as to show the ultimate power of the artist over History and place.


Ph.D.; Oscar Laguna

Ph.D.; Elise Odinot

Ph.D Alexandra Bisotto

B.Sc Bruno Barea

Ph.D.; Pierre Villeneuve

Ph.D.; Jean-Claude Sigoillot

Ph.D Eric ; Record

Craig B Faulds

Frédéric Fine

Laurence Lesage-Meessen

Dr Jérôme Lecomte
email: jerome.lecomte@cirad.fr

B Craig

Ph.D Frédéric Faulds

Ph.D Laurence Fine

Ph.D.; Anne Lesage-Meessen

Ph.D.; Jérôme Lomascolo

Ph.D Lecomte

Bruno Baréa

Anne Lomascolo

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Keywords: rapeseed and sunflower meals, Aspergillus niger, chlorogenate esterase, feruloyl esterase, sinapic acid, caffeic rapeseed and sunflower meals, Aspergillus niger, chlorogenate esterase, feruloyl esterase, sinapic acid, caffeic acid

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Rapeseed (Brassica napus L.) and sunflower (Helianthus annuus L.) are two of the most important oilseed crops in the world [START_REF] Lomascolo | Rapeseed and sunflower meal: a review on biotechnology status and challenges[END_REF] and their oils are mainly used for human consumption or for biofuel production [START_REF] Borredon | Débouchés actuels et futures du tournesol produit en France-Critères de qualité[END_REF][START_REF] Fine | Food losses and waste in the French oilcrops sector[END_REF]. Rapeseed (RSM) and sunflower (SFM) meals are the main coproducts of the seed pressing and de-oiling processes. In 2018, worldwide production of RSM and SFM was estimated at 39.2 Mt and 21.0 Mt respectively (http://www.indexmundi.com/agriculture/). They are mostly dedicated to animal feed because of their high protein content with about 38 g/100 g dry matter (DM) and 28 g/100 g DM for RSM and SFM respectively [START_REF] González-Pérez | Sunflower proteins: overview of their physicochemical, structural and functional properties[END_REF][START_REF] Ivanova | Biochemical characterization of industrially produced rapeseed meal as a protein source in food industry[END_REF][START_REF] Ugolini | Production of an enzymatic protein hydrolyzate from defatted sunflower seed meal for potential application as a plant biostimulant[END_REF]. In addition, studies have shown that these raw materials can also serve as a source of protein isolates with bioactive properties, such as Angiotensin-I Converting Enzyme (ACE) inhibitory activities, antioxidant or antithrombotic activities, and bile acid-binding capacities (Aider and Barbana, 2011;[START_REF] Campbell | Canola/rapeseed protein: future opportunities and directions-workshop proceedings of IRC 2015[END_REF][START_REF] Ugolini | Production of an enzymatic protein hydrolyzate from defatted sunflower seed meal for potential application as a plant biostimulant[END_REF]. These protein isolates also possess emulsification and foaming properties and are considered as promising ingredients for industrial non-food applications such as adhesives and detergents, among others [START_REF] Fetzer | Influence of process conditions during aqueous protein extraction upon yield from pre-pressed and cold-pressed rapeseed press cake[END_REF][START_REF] Fetzer | Rapeseed protein concentrates for non-food applications prepared from pre-pressed and cold-pressed press cake via acidic precipitation and ultrafiltration[END_REF]. Moreover, RSM and SFM can also be used as substrates in microbial fermentative processes like saccharification and production of enzymes, antibiotics and other bioproducts (for a review, see [START_REF] Lomascolo | Rapeseed and sunflower meal: a review on biotechnology status and challenges[END_REF]. Also, as outlined in the work of Licata et al. (2019), these meals represent a potential source of energy production through combustion processes or biogas production and could be considered as economically convenient in a short agro-energy chain.

RSM and SFM also have relatively high total phenolic compound (TPC) contents, ranging from 1-2 g/100 g DM for RSM and 2-4 g/100 g DM for SFM. These phenolics are almost exclusively sinapic acid esters (SAEsters) in RSM, with sinapine as the main derivative (80 % of the TPC content) and the remaining 20 % composed of mono-, diand tri-sinapoyl esters of sugars and/or kaempferol [START_REF] Baumert | Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?[END_REF][START_REF] Milkowski | Sinapate esters in brassicaceous plants: biochemistry, molecular biology, evolution and metabolic engineering[END_REF][START_REF] Siger | Identification and antioxidant activity of sinapic acid derivatives in Brassica napus L. seed meal extracts: main phenolic compounds in rapeseed[END_REF]. In SFM, phenolics are mostly found as caffeic acid esters (CAEsters). 5-Caffeoylquinic acid (chlorogenic acid) is the major phenolic compound accounting for 70 % of the TPC content, while the remaining 30 % are other mono-and di-caffeoylquinic acids, as well as coumaroyl-and feruloylquinic acids. Free caffeic (CA) acid is also found in SFM but accounts for just 0.5 % of the TPC content [START_REF] Guo | A review of phytochemistry, metabolite changes, and medicinal uses of the common sunflower seed and sprouts (Helianthus annuus L.)[END_REF][START_REF] Szydłowska-Czerniak | Optimization of extraction conditions of antioxidants from sunflower shells (Helianthus annuus L.) before and after enzymatic treatment[END_REF][START_REF] Weisz | Identification and quantification of phenolic compounds from sunflower (Helianthus annuus L.) kernels and shells by HPLC-DAD/ESI-MSn[END_REF].

Phenolic acids, particularly hydroxycinnamic acids, are bioactive molecules with interesting properties that include antioxidant [START_REF] Gullón | Yerba mate waste: a sustainable resource of antioxidant compounds[END_REF][START_REF] Shahidi | Phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects -A review[END_REF], anti-inflammatory [START_REF] Oueslati | Phenolic content, antioxidant, anti-inflammatory and anticancer activities of the edible halophyte Suaeda fruticosa Forssk[END_REF] and antimicrobial [START_REF] Ouerghemmi | Antioxidant and antimicrobial phenolic compounds from extracts of cultivated and wild-grown Tunisian Ruta chalepensis[END_REF] activities among others. These bioactive properties can be enhanced by modifying the hydrophilic/hydrophobic balance of the molecules [START_REF] Laguerre | How to boost antioxidants by lipophilization?[END_REF][START_REF] Lecomte | Synthesis, characterization and free radical scavenging properties of rosmarinic acid fatty esters[END_REF][START_REF] Szydłowska-Czerniak | Synthesis of novel octyl sinapate to enhance antioxidant capacity of rapeseed-linseed oil mixture: octyl sinapate as a new lipophilic antioxidant[END_REF]. For example, esterification with aliphatic alcohols of adequate chain-length improved their antioxidant activity in liposome suspensions, oil-in-water emulsions [START_REF] Laguerre | Relationship between hydrophobicity and antioxidant ability of "phenolipids" in emulsion: a parabolic effect of the chain length of rosmarinate esters[END_REF][START_REF] Sørensen | Alkyl chain length impacts the antioxidative effect of lipophilized ferulic acid in fish oil enriched milk[END_REF] and even in cultured cells, as well as their bactericidal activity on selected bacteria [START_REF] Suriyarak | Impact of fatty acid chain length of rosmarinate esters on their antimicrobial activity against Staphylococcus carnosus LTH1502 and Escherichia coli K-12 LTH4263[END_REF]. This phenomenon, related to the so-called "cut-off effect", was attributed to a better location and concentration of esters at the interfacial membrane where lipid oxidation occurs [START_REF] Laguerre | Does hydrophobicity always enhance antioxidant drugs? A cut-off effect of the chain length of functionalized chlorogenate esters on ROS-overexpressing fibroblasts: cut-off effect of phenolipid antioxidants[END_REF][START_REF] Mcclements | Interfacial antioxidants: a review of natural and synthetic emulsifiers and coemulsifiers that can inhibit lipid oxidation[END_REF], and to their ability to cross or interact with membranes of living cells and microorganisms. Moreover, owing to their particular structure and reactivity, phenolic acids (i.e. hydroxybenzoic and hydroxycinnamic acids), as their corresponding vinyl phenols, have gained much importance during the last decade for the synthesis of new sustainable polymers and pre-polymers [START_REF] Aouf | Chemoenzymatic functionalization of gallic and vanillic acids: synthesis of bio-based epoxy resins prepolymers[END_REF][START_REF] Diot-Néant | Biocatalytic synthesis and polymerization via ROMP of new biobased phenolic monomers: a greener process toward sustainable antioxidant polymers[END_REF][START_REF] Pion | Renewable alternating aliphatic-aromatic copolyesters derived from biobased ferulic acid, diols, and diacids: sustainable polymers with tunable thermal properties[END_REF][START_REF] Reano | Structure-activity relationships and structural dsign optimization of a series of p -hydroxycinnamic acids-based bis-and trisphenols as novel sustainable antiradical/antioxidant additives[END_REF][START_REF] Wang | Synthesis of welldefined hyperbranched polymers bio-based on multifunctional phenolic acids and their structure-thermal property relationships[END_REF]Zago et al., 2015a). So any raw material and process enabling large-scale production and recovery of phenolic acids would positively facilitate the development of these sustainable active platform molecules and polymers. In this context, SFM and RSM, which are cheap, abundant, and rich in phenolics, make perfectly suitable candidates. It is worth noting that only few studies have reported the use of these two raw materials for the production of phenolic acids and derivatives like vinyl phenols [START_REF] Kreps | Industrial rapeseed and sunflower meal as source of antioxidants[END_REF][START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF].

Microbial cinnamoyl and feruloyl esterases are carboxylic ester hydrolase enzymes (EC 3.1.1) that are able to hydrolyze ester linkages between hydroxycinnamic acids and sugars present in plant cell-walls [START_REF] Faulds | What can feruloyl esterases do for us?[END_REF] with various in vivo specificities according to type of enzyme and origin of microorganism. For instance, the type-A feruloyl esterase from the filamentous fungus Aspergillus niger (called AnFaeA) is highly active on sinapic and ferulic acid methyl esters and on some 1,5-esterified feruloylated oligomers from cereals. Type-B feruloyl esterase from A. niger (so called AnFaeB) is rather active on caffeic and p-coumaric acid methyl esters and on some 1,2and 1,6-esterified feruloylated oligomers from sugar-beet pulp [START_REF] Brézillon | Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar-beet pulp[END_REF][START_REF] Faulds | Purification and characterization of a ferulic acid esterase (FAE-III) from Aspergillus niger: specificity for the phenolic moiety and binding to microcrystalIine cellulose[END_REF]. Another type of A. niger carboxylic ester hydrolase, called chlorogenic acid hydrolase (or chlorogenate esterase, ChlE) was primarily found to be highly specific to chlorogenic acid (5-O-caffeoylquinic acid) hydrolysis [START_REF] Benoit | Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger[END_REF]. Historically, pure AnFaes were shown to be essentially inactive when used directly on natural raw substrates. However, we recently studied the enzymatic hydrolysis of an industrial rapeseed meal (I-RSM) using a recombinant AnFaeA to produce sinapic acid [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF] and demonstrated that this enzyme was highly effective on the raw RSM. In the best conditions, AnFaeA hydrolyzed most of the SAEsters present in the RSM, with an overall yield of 68-76 % (100% hydrolysis of sinapine).

Taking this previous work forward, here we aimed to find the best route and operating conditions for producing free CA and SA by applying various fungal carboxylic ester hydrolases from recombinant Aspergillus niger strains either directly on crude meal or on their phenolic extracts obtained by methanolic extraction. Two types of meals were studied: (i) I-meals (industrial meals), produced by a process that includes cooking at 95-100°C and steam desolventizing at 105-107°C, and (ii) NI-meals (custom-made non-industrial meals), obtained at pilot-scale with much milder heat treatment. In a general way, the NI-meals have a net higher phenolic content than the corresponding I-meals (Zago et al., 2015b). We first studied the hydrolysis of sunflower meals (NI-SFM and I-SFM) with two types of fungal ester hydrolases and determined the best conditions for caffeic acid (CA) production. Exploiting their hydrolytic activity on CAEsters [START_REF] Faulds | What can feruloyl esterases do for us?[END_REF][START_REF] Gopalan | Review on technological and scientific aspects of feruloyl esterases: A versatile enzyme for biorefining of biomass[END_REF][START_REF] Ramos-De-La-Peña | Methods and substrates for feruloyl esterase activity detection, a review[END_REF], we tested an A. niger type-B recombinant feruloyl esterase (AnFaeB) and a recombinant A. niger chlorogenate esterase (ChlE) on the SFMs. To the best of our knowledge, these two enzymes have never been used on oilseed meals. Enzymatic hydrolysis was also performed on methanolic extracts from NI-SFM which are mainly composed of phenolics, minerals, and carbohydrates [START_REF] Sripad | Effect of methods to remove polyphenols from sunflower meal on the physicochemical properties of the proteins[END_REF] but are devoid of the proteins and other meal constituents. Then, the same approach was applied to a non-industrial rapeseed meal (NI-RSM) and the corresponding phenolic extract, by using AnFaeA to release free SA. The results (hydrolysis yield and highest released SA amount) were compared with our previous data obtained on an I-RSM [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF]. All cases were compared and discussed based on the following parameters: hydrolysis yields, qualitative and quantitative composition of hydrolyzed hydroxycinnamoyl esters, and free CA or SA amount released per gram of substrate.

Materials and Methods

Chemicals

Sinapine thiocyanate (99.0 % HPLC) was isolated from rapeseed meal according to the method outlined by [START_REF] Mailer | Anti-nutritional components, fibre, sinapine and glucosinolate content, in Australian canola (Brassica napus L.) meal[END_REF]. 3,4-di-O-caffeoylquinic, 3,5-di-Ocaffeoylquinic and 4,5-di-O-caffeoylquinic acids (>99.0 % HPLC) were isolated from yerba mate leaves, following the procedure previously described in [START_REF] Tong | Dicaffeoylquinic acid-enriched fraction of Cichorium glandulosum seeds attenuates experimental type 1 diabetes via multipathway protection[END_REF].

Sinapic acid (98 %) was from AlfaAesar (Karlsruhe, Germany). 4-and 5-caffeoylquinic (chlorogenic) acids (95 %), caffeic acid (>98 %), methanol and water (both HPLC grade, >99.9 %), NaOH (> 97%) and MOPS (3-(N-morpholino)propanesulfonic acid) buffer were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France Five or six successive immersion-filtration steps were required to remove the oil from the SFM and RSM, respectively. The defatted meals were then desolventized in the same device, under vacuum at 60°C and without steam injection. Then, the meals obtained were finely ground by a high-shear impact mill (Hosokawa-alpine, type UPZ100, Augsburg, Germany) using a 0.1-mm (for NI-RSM) or a 0.5-mm (for NI-SFM) grid operated at room temperature at a speed of 18,000 rpm, and a feeder speed of 4 kg.h -1 .

Industrial sunflower and rapeseed meals (I-SFM and I-RSM)

I-SFM and I-RSM were provided by the Technical Centre for Oilseed Crops, Grain Legumes and Industrial Hemp (Terres Inovia, Pessac, France). I-SFM and I-RSM were obtained from seed pressing and hexane solvent oil extraction. Further industrial processing steps included: preconditioning at about 45°C, cooking at 95-100°C for 60 min, then steam desolventizing at 107 ± 2°C for 80 ± 5 min in the case of I-RSM or 105 ± 2°C for 50 ± 5 min in the case of I-SFM.

Enzyme production

The extracellular recombinant feruloyl esterase A (AnFaeA), feruloyl esterase B (AnFaeB) and chlorogenate esterase (ChlE) were produced, respectively, by the recombinant strains A. niger BRFM451, A. niger BRFM766 and A. niger BRFM891, formerly obtained in our Laboratory of Fungal Biodiversity and Biotechnology (UMR1163 BBF, Marseille, France), by genetic engineering of the host strain A. niger D15#26, as previously described [START_REF] Benoit | Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger[END_REF][START_REF] Levasseur | Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme[END_REF][START_REF] Record | Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application[END_REF]. Briefly, in this study, these strains were used to produce batches of AnFaeA, AnFaeB and ChlE, respectively, in a culture medium containing (per liter contents):

glucose 50 g, NaNO3 5.95 g, KCl 0.52 g, MgSO4 [START_REF] Levasseur | Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme[END_REF].

Accession number of protein sequences

The protein sequences of AnFaeA, AnFaeB and ChlE were registered in the NCBI database under accession numbers CAA70510, AJ309807, and ABK62698, respectively.

Enzyme activity assay

Esterase activity was assayed spectrophotometrically at either 37°C and 55°C for AnFaeA or 37°C and 50°C for AnFaeB and ChlE, as previously described [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF], by monitoring the A335 with respect to the rate of hydrolysis of 0.032 mM of the enzyme substrate in 88 mM of a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 5.5 for AnFaeA and pH6 for AnFaeB and ChlE). The synthetic substrates used were methyl sinapate for AnFaeA, methyl caffeate and methyl p-coumarate for AnFaeB, and chlorogenic acid for ChlE. The extinction coefficients at 335 nm were (in L.mol -1 .cm -1 ): 13,318 for methyl sinapate, 5,500 for sinapic acid, 19,524 for methyl ferulate, 4,409 for ferulic acid, 12,560 for methyl caffeate, 6,060 for caffeic acid, 821

for methyl p-coumarate, 673 for p-coumaric acid and 15,423 for chlorogenic acid.

Endogenous polyphenol oxidase (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1) activity was determined in SFM as per a procedure adapted from [START_REF] Halaouli | Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications[END_REF]. Five grams of meal were homogenized in 50 mL sodium phosphate buffer (100 mM, pH 6.8) at 37°C for 3h under stirring at 200 rpm. After centrifugation at 9500

× g for 40 min, the supernatant was used to assay monophenol oxidase (or cresolase)

and diphenol oxidase (or catecholase) activity [START_REF] Halaouli | Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications[END_REF]. Briefly, monophenolase activity was determined in meal extract by monitoring the A484 (extinction coefficient 22,300 L.mol -1 .cm -1 ) with respect to rate of oxidation of 1.25 mM L-tyrosine in sodium phosphate buffer (50 mM, pH6.8) at 30°C, in the presence of 3methyl-2-benzothiazolinonehydrazone (MBTH, 5 mM) and L-DOPA using a molar L-DOPA-to-L-tyrosine ratio of 0.057. Diphenolase activity was determined by monitoring A484 (extinction coefficient 22,300 L.mol -1 .cm -1 ) with respect to rate of oxidation of 4.2 mM L-DOPA in sodium phosphate buffer (50 mM, pH6.8) at 30°C, in the presence of 5 mM MBTH.

All enzymatic activities were expressed in nanokatals. One nanokatal of activity is defined as the quantity of enzyme that hydrolyzed 1 nmol of substrate per second.

Experiments were performed in triplicate, and the standard deviation was lower than 1% of the mean.

Determination of meal dry matter and lipid contents

Dry matter content of the meals was determined as follow: around two grams (exactly weighed at +/-0.01 mg) of meal were placed into an aluminium capsule (previously weighed dry with the same precision scale) and the whole was dried at 105 °C until constant weight (difference between two weighings < 1%). Then, capsule and meal were cooled in a desiccator at room temperature for 30 min and finally weighed.

The dried matter content was calculated from the difference between the weight of the sample (meal and capsule) before and after drying to constant weight.

Lipid content of the meals was determined by the method previously outlined in the work of [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF] : Around two grams (exactly weighed at +/-0.01 mg)

of each fraction were placed into a filter paper envelope previously dried and weighed and the whole was dried at 105 °C until constant weight. Then, envelopes were extracted with n-hexane for 5 h using a Soxhlet apparatus. After extraction, envelopes were desolventized at room temperature for 12 hours, dried at 105 °C, then cooled in a desiccator at room temperature for 30 min and finally weighed. The oil content was calculated from the difference between the dry weight of the sample (meal and envelope) before and after defatting. Values were given as percentage of oil per gram of dry matter.

These two values were taken into account to express other results per gram of dry defatted matter. All experiments were done in triplicate.

Methanolic extraction of phenolic compounds from meals

The extraction of TPC from meal samples was carried out in closed tubes at 75°C for 20 min using a meal-to-methanol ratio of 1:100 (w/v) following the procedure previously described in [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF]. After drying under a nitrogen stream, extracts were kept at -20°C until being processed. All experiments were done in triplicate.

Determination of the potentially releasable phenolic acid content in meals by alkaline hydrolysis

Fifty milligrams of meal were mixed with 1.5 mL of methanol. Then, 6 mL of 2 M NaOH were added to the mixture and incubated at 30°C for different times (15, 30, 60 and 120 min). The mixture was then acidified to pH 2.0 using 4 M HCl with addition of 300 mg NaCl and further extracted three times with ethyl acetate (2 mL) by stirring for 2 min at room temperature. After each extraction, samples were centrifugated (3000 × g, 5 min) and supernatants were collected and pooled. The organic phase was dried under nitrogen stream and the residue was kept at -20°C until HPLC analysis. All experiments were performed in triplicate. The amount of free phenolic acid released from meals after alkaline hydrolysis was used, when possible, as the 100% baseline reference for the calculations of enzymatic hydrolysis yields.

Enzymatic hydrolysis of caffeic acid derivatives in sunflower meals and in the corresponding phenolic extracts with AnFaeB and ChlE

Fifty milligrams of the meal were weighed into a brown flask with a screw cap, and 5 mL of 100 mM MOPS buffer at pH 6 were added. The closed flasks were incubated in an orbital shaker (350 rpm) at 50°C run for 30 min for homogenization.

After this pre-incubation step, two samples (mixture of meal and MOPS buffer) were cooled to -80°C and freeze-dried. Phenolics were then extracted as described in section 2.7, and their content determined and set as t = 0. In parallel, different amounts of ChlE and AnFaeB, from 0 to 30 nkat enzyme/g meal, were added to other pre-incubated samples. The hydrolysis assay was run for 10 to 240 min, at 50°C and 350 rpm. An enzyme-free control was also performed in the same conditions. After the desired treatment duration, the reaction mixture was cooled to -80°C and freeze-dried.

Phenolics were then extracted as described in section 2.7. After drying under a nitrogen stream, extracts were kept at -20 °C until HPLC analysis. Two independent trials were performed per experiment.

The enzymatic treatment was also applied to a dry methanolic extract obtained from 50 mg of NI-SFM as described in section 2.7. Hydrolysis was performed at 50°C, as described above, on the dry phenolic extract solubilized in 5 mL MOPS buffer containing 30 nkat/g meal for AnFaeB or 10 nkat/g meal for ChlE, respectively. After treatment, the reaction medium was cooled to -80°C, then freeze-dried and finally extracted as described in section 2.7. After drying under a nitrogen stream, the dry residue was kept at -20°C until HPLC analysis. Two independent trials were performed per experiment.

Enzymatic hydrolysis of sinapic acid derivatives in rapeseed meal and in the corresponding phenolic extracts with AnFaeA

The enzymatic hydrolysis of NI-RSM with AnFaeA was performed in the optimized conditions previously determined on I-RSM by [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF], with some modifications. Briefly, 50 mg of the meal was weighed into a brown flask with a screw cap and 5 mL of 100 mM MOPS buffer at pH 5.5 was added. The closed flasks were stirred in an orbital shaker (350 rpm) at 55°C run for 30 min for homogenization.

After this pre-incubation step, two samples (mixture of meal and MOPS buffer) were cooled to -80°C and freeze-dried. Phenolics were then extracted as described in section 2.7 and their content determined and set as t = 0. In parallel, 39 nkat AnFaeA/g meal was added to other pre-incubated samples. The enzymatic hydrolysis assay was run at 55°C, 350 rpm for 10 to 240 min. An enzyme-free control was also performed in the same conditions. After treatment, the reaction mixture was cooled to -80°C and the mixture was freeze-dried. The methanolic extraction of phenolics was done as described in section 2.7. After drying under a nitrogen stream, extracts were kept at -20°C until HPLC analysis. Two independent trials were performed per experiment. Enzymatic hydrolysis was also applied to a dry NI-RSM methanolic extract obtained from 50 mg of meal as described in section 2.7. Hydrolysis was performed at 55°C, as described above, on this dry phenolic extract solubilized in 5 mL MOPS buffer containing 39 nkat AnFaeA/g meal. After treatment, the reaction medium was cooled to -80°C, then freezedried and finally extracted as described in section 2.7. After drying under a nitrogen stream, the dry residue was kept at -20°C until HPLC analysis. Two independent trials were performed per experiment.

High Performance Liquid Chromatography (HPLC) analysis of phenolics from hydrolyzed meals

The dry phenolic extracts, obtained as described in the previous sections, were solubilized in 5 mL methanol/water (2/1, v/v) and filtered (0.45-μm nylon filter) before HPLC analysis. Phenolic compounds were quantified with an XR UFLC Shimadzu liquid-phase chromatograph equipped with an SPD-M20A diode array detector (Kyoto, Japan). Separation was carried out with an ACE C18 reversed-phase column (5 μm, 250 mm × 4.6 mm, Phenomenex, Le Pecq, France). Injection volume, flow rate, and oven temperature were set at 20 μL, 1 mL.min -1 and 30°C respectively. The gradient elution was performed using HPLC-grade water with 0.1% (v/v) acetic acid (A) and methanol with 0.1% (v/v) acetic acid (B) in the following conditions: 15% B (0-5 min), 15-80% B (5-30 min), 80-100% B (30-31 min), 100% B (31-35 min), 100-15% B (35-40 min).

Sunflower samples

Sunflower phenolics were identified according to retention times and quantified at their maximum detection wavelength, as previously described in [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF]. Total caffeic acid derivative (TCAD) content was calculated by summing peak area of all caffeoyl-containing molecules after normalization (i.e. each peak area was divided by the number of caffeoyl moiety contained in the corresponding molecule) and expressed in µmol of caffeic acid equivalent (CAE)/g DDM. CA was selected as external standard and the calibration curve was obtained from series of standard methanolic solutions from 12.5 to 800 µM (R 2 = 0.9991).

Rapeseed samples

SA and sinapine thiocyanate were chosen as external standards for the calibration curves. Their maximum detection wavelength and retention time were 323 nm-17.8 min and 328 nm-11.5 min, respectively. Total sinapic acid derivative (TSAD) content was calculated by summing peak area of all sinapoyl-containing molecules after normalization (i.e. each peak area was divided by the number of sinapoyl moiety in the corresponding molecule) and expressed in µmol of sinapic acid equivalent (SAE)/g DDM. Calibration curves were obtained from series of standard methanolic solutions from 12.5 to 200 µM with R 2 = 0.9983 for sinapic acid and R 2 = 0.9987 for sinapine.

Statistical analyses

Means and standard deviations (SD) were calculated using Minitab Statistical Software v.18. The means were then compared using one-way ANOVA analysis, with level of significance set to  = 0.05.

Results and discussion

Characterization of raw materials

Moisture, lipid and total phenolic compound contents of the meals

The moisture and lipid contents of the meals used in this study are shown in Table 1, with values of 7-13% DM and 1.4-1.9% DM, respectively. These data were consistent with the values available in the literature [START_REF] Lomascolo | Rapeseed and sunflower meal: a review on biotechnology status and challenges[END_REF].

Table 1 (suggested location)

The TPC initially present in SFM and RSM samples, as determined after methanolic extraction, are shown in Table 2 and Table 3 respectively. The TPC content of SFM samples, which was expressed as TCAD content as 95 % of SFM phenolics were found as caffeic acid esters [START_REF] Karamać | Antioxidant activity of phenolic compounds identified in sunflower seeds[END_REF][START_REF] Weisz | Identification and quantification of phenolic compounds from sunflower (Helianthus annuus L.) kernels and shells by HPLC-DAD/ESI-MSn[END_REF], was 71.7  0.5 µmol TCAD/g DDM for NI-SFM and 57.4  1.5 µmol TCAD/g DDM for I-SFM. These data were consistent with the values available in the literature [START_REF] Baumert | Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?[END_REF][START_REF] Cai | A rapid high-performance liquid chromatographic method for the determination of sinapine and sinapic acid in canola seed and meal[END_REF][START_REF] Weisz | Identification and quantification of phenolic compounds from sunflower (Helianthus annuus L.) kernels and shells by HPLC-DAD/ESI-MSn[END_REF]. The TPC content of RSM samples, which was expressed as TSAD content as 98 % of their phenolics were shown to be sinapic acid esters [START_REF] Khattab | Determination of sinapic acid derivatives in canola extracts using high-performance liquid chromatography[END_REF][START_REF] Siger | Identification and antioxidant activity of sinapic acid derivatives in Brassica napus L. seed meal extracts: main phenolic compounds in rapeseed[END_REF], was 64.2  1.2 µmol TSAD/g DDM for NI-RSM and 44.8  0.2 µmol TSAD/g DDM for I-RSM. Thus, TPC content was 1.2-to 1.4-fold higher in NI-meals than I-meals, in our conditions.

A mild alkaline treatment (NaOH 2 M, 30°C, 30 min) was applied to NI-SFM and I-SFM samples in order to determine their respective total releasable CA content (CAEsters in soluble form and potentially bound to the lignocellulosic matrix [START_REF] Shahidi | Insoluble-bound phenolics in food[END_REF])), to be taken as reference. Unfortunately, no CA was recovered (Table 2) from either NI-SFM or I-SFM despite the mild alkaline treatment used. Indeed, the high chemical reactivity of CA make it prone to autoxidation at high pH due to its catechol function that rapidly reacts with NaOH and oxidizes in the presence of reactive oxygen species [START_REF] Wildermuth | Chlorogenic acid oxidation and its reaction with sunflower proteins to form green-colored complexes: chlorogenic acid oxidation[END_REF]. Thus, as alkaline hydrolysis was not suitable for assessing the total potentially releasable CA from SFM, all calculations and comparisons between various enzymatic hydrolysis conditions were made on the basis of TCAD content after methanolic extraction, expressed as µmol CAE/g DDM, and

determined from the values of 71.7 ± 0.5 µmol CAE/g DDM for NI-SFM and 57.4 ± 1.5 µmol CAE/g DDM for I-SFM (Table 2). These values were taken as 100% reference for the calculations of enzymatic hydrolysis yields.

Table 2 (suggested location)

As done for SFM samples, the total amount of SA initially present in rapeseed meals as soluble conjugates and potentially releasable forms was determined after alkaline hydrolysis. The result was 62.5 ± 1.8 µmol/g DDM for NI-RSM and 46.8 ± 3.0 µmol/g DDM for I-RSM (Table 3). These values were not significantly different from those of the initial TSAD content of each meal (64.2 ± 1.2 µmol SAE/g DDM for NI-RSM and 44.8 ± 0.2 µmol SAE/g DDM for I-RSM), indicating that all the sinapic esters from the meal were hydrolyzed and recovered after our alkaline hydrolysis protocol. The latter values, which represented the total amount of potentially releasable SA per gram of DDM, were set as the 100% reference and used for the calculations of enzymatic hydrolysis yields. Unlike the significant loss of phenolics observed by Vuorela et al.

after alkaline hydrolysis of RSM with a 4 M NaOH solution at 37°C for 4 h [START_REF] Vuorela | Quantitative analysis of the main phenolics in rapeseed meal and oils processed differently using enzymatic hydrolysis and HPLC[END_REF], the milder conditions (NaOH 2 M, 30°C, 30 min) implemented here managed to not degrade the SA and its derivatives.

Table 3 (suggested location)

Characterization of meal phenolic composition

This study identified the phenolic compounds present in the I-SFM by HPLC-DAD. As shown by HPLC analysis of I-SFM methanolic extract (Fig. 1), the main phenolic compounds were CAEsters such as mono-and di-caffeoylquinic acids, the most abundant being 4-O-caffeoylquinic acid (4-CQA) and 5-O-caffeoylquinic acid (5-CQA). These two compounds co-eluted and were named chlorogenic acid. Other minor phenolics were 3-O-caffeoylquinic acid (3-CQA), two di-caffeoylquinic acid (di-CQA) isomers (3,5-and 4,5-di-O-caffeoylquinic acids), 5-O-p-coumaroylquinic acid, 5-Oferuloylquinic acid, and CA. These results were in good agreement with other studies [START_REF] Karamać | Antioxidant activity of phenolic compounds identified in sunflower seeds[END_REF][START_REF] Weisz | Identification and quantification of phenolic compounds from sunflower (Helianthus annuus L.) kernels and shells by HPLC-DAD/ESI-MSn[END_REF], and were comparable to the NI-SFM composition previously determined by [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF].

Figure 1 (suggested location)

Two of our previous studies characterizing the phenolic composition of NI-RSM and I-RSM [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF][START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF] showed that RSM phenolics comprised: sinapine, sinapoyl glucopyranoside, sinapic acid, di-and tri-sinapoyl gentiobiosides, disinapoyl glucopyranoside isomer, and sinapoyl keampferol derivatives.

Release of free caffeic acid from sunflower meals by A. niger AnFaeB and ChlE

Enzymatic hydrolysis of I-SFM

This study selected AnFaeB and ChlE for the hydrolysis of phenolic esters present in SFMs. Numerous studies have already shown that these enzymes can hydrolyze CAEsters in other raw materials such as coffee pulp, apple marc, autoclaved maize bran and sugar-beet pulp [START_REF] Benoit | Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products[END_REF][START_REF] Benoit | Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger[END_REF][START_REF] Faulds | What can feruloyl esterases do for us?[END_REF][START_REF] Gopalan | Review on technological and scientific aspects of feruloyl esterases: A versatile enzyme for biorefining of biomass[END_REF]; Ramos-de-la-Peña and Contreras-Esquivel, 2016), but SFM has never yet been investigated. The optimum pH and temperature of these enzymes established in previous work were set to 6.0 and 50°C for synthetic substrates (e.g.

methyl sinapate and 5-CQA) [START_REF] Asther | Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalysing the hydrolysis of chlorogenic acid[END_REF][START_REF] Benoit | Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products[END_REF].

Given the drastic heat treatment undergone during the production process, it is reasonable to posit that I-SFM was somewhat recalcitrant to enzymatic degradation. We therefore traced the kinetics of hydrolysis of the major phenolic esters in I-SFM and the release of free CA as a function of enzyme amount (Fig. 2). In the presence of 20 to 30 nkat AnFaeB/g meal, all the CAEesters, initially present in I-SFM, were completely hydrolyzed after 1 h of incubation (Fig. 2, Supplementary data Fig. S1). An amount of 5 nkat AnFaeB/g meal failed to completely hydrolyze the phenolics. An amount of 10 nkat AnFaeB/g meal was able to hydrolyze all CAEesters except 3-CQA (Fig. 2). A minimum concentration of 20 nkat AnFaeB/g meal was thus required for complete hydrolysis of all phenolics, and the highest free CA content was obtained with 30 nkat AnFaeB/g meal after a 3 h incubation (59.8 ± 2.1 µmol/g DDM), which corresponded to a 100% hydrolysis yield. AnFaeB thus showed high catalytic efficiency on all the mono-and dicaffeoylquinic acid isomers present in I-SFM (Fig. 2), together with the pcoumaroyl and feruloyl derivatives (Supplementary data, Fig. S1).

Figure 2 (suggested location)

In the presence of ChlE, the hydrolysis of 3,5-diCQA and 4,5-diCQA was complete after 3 h of incubation whatever the amount of enzyme. Conversely, 3-CQA was not hydrolyzed at all in any condition, even though 4-CQA and 5-CQA were 90% hydrolyzed after 2 h of incubation with ChlE (Fig. 2). The highest free CA content of obtained from I-SFM with ChlE was 54.0 ± 1.1 µmol/g DDM after 2-4 h incubation with 5-30 nkat enzyme/g meal. Compared to AnFaeB, this slightly lower value of free CA released was mainly due to the inability of ChlE to hydrolyze 3-CQA, which was still found at 6.1 ± 0.3 µmol CAE/g DDM whatever the enzyme concentration (Fig. 2, supplementary data Fig. S1).

AnFaeB has already been shown to have a broader specificity than ChlE for hydrolyzing chlorogenic acids from agricultural by-products such as maize bran and sugar-beet pulp [START_REF] Benoit | Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products[END_REF]. For instance, unlike ChlE, AnFaeB was found to hydrolyze 5-O-p-coumaroylquinic acid and feruloylated oligomers [START_REF] Benoit | Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger[END_REF].

This confirmed the decisive role of the moieties adjacent to the ester bond for correct alignment of the substrate in the active site of Faes. To the best of our knowledge, there is no literature data on the relative activity of AnFaeB and ChlE against the different mono-and di-caffeoylquinic acids. Here, we demonstrated that ChlE showed selectivity for different caffeoylquinic acid isomers in the SFM compared to AnFaeB. The concentrations of 30 nkat AnFaeB/g meal and 10 nkat ChlE/g meal were then chosen to be further applied on NI-SFM

Enzymatic hydrolysis of NI-SFM

Fig. 3A and Fig. 3B shows the results of enzymatic hydrolysis performed on NI-SFM with AnFaeB and ChlE, respectively. When AnFaeB was used, all the chlorogenic acid (4-and 5-CQA) was hydrolyzed after 10 min. When ChlE was used, 4-and 5-CQA were 95% hydrolyzed in our conditions. The highest concentration of free CA released in the reaction medium was observed after 30 min with AnFaeB (52.3 ± 0.2 µmol/g DDM) and ChlE (42.0 ± 1.1 µmol/g DDM). However, these values remained lower than the initial concentration of chlorogenic acid of 55.8 ± 0.9 µmol CAE/g DDM in the meal (Table 2). It is worth noting that there was a strong decrease in TCAD content over time (Fig. 3), with or without enzyme (control), resulting in a loss of TCAD compared to the initial content in the meal (Table 2). While the effect of other factors (including pH and temperature) cannot be totally ruled out, the most likely explanation was the presence of endogenous enzymes in the NI-SFM that degraded CA and its derivatives during the pre-incubation and incubation steps. Studies have already demonstrated the presence of polyphenol oxidase (PPO) in sunflower meal and its high activity on chlorogenic acid [START_REF] Wildermuth | Chlorogenic acid oxidation and its reaction with sunflower proteins to form green-colored complexes: chlorogenic acid oxidation[END_REF]. Therefore, although our hydrolysis conditions (pH 6.0, 50°C) did not exactly correspond to PPO optimum pH and temperature (7.5 and 45°C respectively; [START_REF] Wildermuth | Chlorogenic acid oxidation and its reaction with sunflower proteins to form green-colored complexes: chlorogenic acid oxidation[END_REF], we found it reasonable to posit that the residual PPO activity was sufficient to continuously oxidize chlorogenic acid and related compounds from the pre-incubation step. Here we measured PPO activity in endogenous SFM meal extracts and found 1.02 ( 0.08) nkat/mL diphenol oxidase activity and 0.43 (0.02) nkat/mL monophenol oxidase activity, which are characteristic of a tyrosinase-type activity [START_REF] Halaouli | Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications[END_REF]. In addition, it is noteworthy that 15 putative PPO-encoding gene sequences were predicted in the publicly-available Helianthus annuus genome from the NCBI Database (https://www.ncbi.nlm.nih.gov/search/all/?term=polyphenol+oxidase+helianthus+annuu s). Thus, it seemed that the heat treatment (cooking at 95-100°C followed by steam desolventizing at about 105°C) applied to I-SFM, but not NI-SFM, led to inactivation of endogenous enzymes in the SFM even if this heat treatment was also responsible for a significant decrease in TPC (Table 2). This constituted a major drawback that greatly limited the value of non-thermally-treated NI-SFM as a substrate for in situ enzymatic production of CA.

AnFaeB and ChlE hydrolysis of NI-SFM methanolic extract

Enzymatic hydrolysis was also performed on phenolic extracts from NI-SFM after a sequence of methanolic extraction, drying, and solubilization in reaction buffer.

Methanol mainly extracted phenolic compounds, minerals and carbohydrates from the meal [START_REF] Sripad | Effect of methods to remove polyphenols from sunflower meal on the physicochemical properties of the proteins[END_REF], but not the proteins, including the possible endogenous enzymes responsible for the degradation of phenolics. Fig. 4A shows the fast hydrolysis of CAEsters by AnFaeB in the methanolic extract from NI-SFM at 50°C.

After only 10 min, all the phenolic esters were hydrolyzed into free CA, which gave rise to a concentration of 68.1 ± 1.7 µmol/g DDM with 95 % hydrolysis yield (Table 4).

Figure 4 (suggested location) When hydrolysis was carried out with ChlE at the same temperature (50°C), chlorogenic acid was hydrolyzed more slowly (30 min instead of 10 min) and was not completely converted even after 240 min (Fig. 4-B), and only a small fraction of 3-CQA got hydrolyzed (Table 4, Supplementary data Fig. S2). This typical feature of ChlE, already observed during I-SFM hydrolysis (see section 3.2.1), could explain why the highest concentration of CA was only 55.4 ± 0.3 µmol/g DDM after 60 min incubation, which corresponded to a hydrolysis yield of only 77 % (Table 4). It is worth noting that, in both cases (AnFaeB and ChlE), no decrease in the TCAD content was observed over enzymatic incubation time on methanolic extracts (Fig. 4), conversely to the crude NI meal (Fig. 3). This might indirectly confirm the presence of phenolic-degrading enzymes (i.e PPO for instance) in the meal that were removed after phenolic extraction with methanol.

On NI-SFM, the best CA yields were achieved through enzymatic hydrolysis of methanolic extract, particularly with AnFaeB that allowed fast, selective, and quantitative production of CA (68.1  1.7 µmol/DDM from methanolic extract hydrolysis against 52.3  0.2 µmol/g DDM from crude NI-meal hydrolysis).

Enzymatic hydrolysis of sinapic acid esters in NI-RSM and in the corresponding methanolic extract with AnFaeA

Hydrolysis conditions (pH, temperature and enzyme concentration) for the enzymatic hydrolysis of sinapic esters present in an I-RSM (a commercial RSM thermally treated by cooking at 95-100°C followed by steam desolventizing at about 107°C) was previously established by [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF]. These conditions were implemented on NI-RSM and on the corresponding methanolic extract. NI-RSM was chosen for its higher total sinapic acid derivatives (TSAD) content compared to the I-RSM (Table 1).

AnFaeA hydrolysis of NI-RSM

The evolution of the concentration of sinapine (SNP), sinapic acid (SA) and TSAD during enzymatic hydrolysis of NI-RSM with AnFaeA (39 nkat/g meal, 55°C, pH 5.5) is charted in Fig. 5A. The maximum amount of SA released was 41.3 ± 0.3 µmol/g DDM after 240 min of incubation with AnFaeA. Almost all the SNP initially present in the meal (31.7 ± 0.6 µmol SAE/g DDM) (Fig. 5A) and the mono-and disinapoyl glucose isomers (Supplementary data Fig. S3A) were hydrolyzed at the end of incubation.

However, kaempferol-based sinapic acid derivatives, as well as di-sinapoyl gentiobiosides and some tri-sinapoyl gentiobiosides were not (or partially) hydrolyzed by the enzyme (supplementary data, Fig. S3A). These results were in agreement with our previous work on I-RSM [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF]. TSAD content measured after the 30min pre-incubation step (Fig. 5A) was decreased by about 16% compared to the initial content in the unprocessed meal (64.2  1.2 µmol SAE/g DDM), possibly due to the activity of endogenous enzymes (possibly PPO) present in the crude meal. In the case of NI-RSM, no PPO-type activity was detectable in the meal in our assay conditions.

However, it is noteworthy that a laccase-encoding gene (AtTT10 gene) was identified in the genome of Brassica napus [START_REF] Zhang | Gene silencing of BnTT10 family genes causes retarded pigmentation and lignin reduction in the seed coat of Brassica napus[END_REF]. This PPO was involved in proanthocyanidin polymerization and lignin biosynthesis as well as seed coat pigmentation. Conversely, this decrease was not observed in enzymatic hydrolysis applied to an I-RSM [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF]. Like for I-SFM, but in contrast to NI-RSM, the thermal treatments (95-107°C) applied to I-RSM seemed to inactivate endogenous phenolic-degrading enzymes. 5), concomitantly with the appearance of a new phenolic compound in the reaction medium (Fig. 5B, green bar) that corresponded to a peak with a retention time of 22.25 min in our HPLC conditions (Supplementary data Fig. S3B).

Table 5 (suggested location)

Based on its retention time in the HPLC chromatogram (Compound 9, Supplementary data Fig. S3B), its maximum wavelength absorption of 326 nm, and its mass and NMR ( 1 H, 13 C) spectra (Supplementary data Fig. S4 and Fig. S5), the new compound was 1,2-di-O-sinapoyl--D-glucose. Therefore, given the presence of significant amounts of glucose [START_REF] Naczk | Carbohydrates of canola and rapeseed[END_REF], as well as mono-and disinapoyl glucose isomers in the NI-RSM extract [START_REF] Laguna | Production of proteins and phenolic compounds enriched fractions from rapeseed and sunflower meals by dry fractionation processes[END_REF], it can be reasonably assumed that this new compound resulted from transesterification of sinapine with the aforementioned molecules extracted from the meal. If so, these results would confirm the well-known acyl transferase activity of AnFaeA [START_REF] Hatzakis | Asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens[END_REF][START_REF] Hüttner | Immobilisation on mesoporous silica and solvent rinsing improve the transesterification abilities of feruloyl esterases from Myceliophthora thermophila[END_REF][START_REF] Vafiadi | Feruloyl esterase-catalysed synthesis of glycerol sinapate using ionic liquids mixtures[END_REF]. However, the reason why such a reaction only occurred in the NI-RSM methanolic extract remains to elucidate.

TSAD content measured during enzymatic hydrolysis of the methanolic extract (59.2 ± 2.3 µmol SAE/DDM) remained higher than TSAD content measured during meal hydrolysis (53.1 ± 1.0 µmol SAE/ DDM). This might corroborate the presence of phenolic-degrading endogenous enzymes in the crude meal.

Hydrolysis of the NI-RSM methanolic extract was also performed at 37°C in order to assess the possible influence of temperature on yield of 1,2-di-O-sinapoyl--Dglucose synthesis (Fig. 5C). At 37°C, TSAD concentration (65.6 ± 2.3 µmol/ DDM) measured during hydrolysis remained stable throughout the incubation time at an equivalent level to the initial content in the unprocessed meal (64.2  1.2 µmol SAE/g DDM). The decrease over time of SNP was slightly slower at 37°C than at 55°C (Fig. 5C): hydrolysis was complete in less than 120 min at 55°C whereas 3% of SNP remained in the medium even after 240 min at 37°C. Production of 1,2-di-O-sinapoyl--D-glucose was higher overall (e.g. 8.0 ± 0.8 µmol SAE/g DDM at 37°C against 5.7 ± 0.3 µmol SAE/g DDM at 55°C after 240 min incubation), which may suggest that lower temperature facilitated acyl transfer more than sinapine hydrolysis, which occurs in both conditions.

Taken together, the results presented in section 3.4 converged to show the best yield of SA came from hydrolysis performed directly in the crude NI-RSM with

AnFaeA compared to hydrolysis of the corresponding methanolic extract. The enzymatic treatment of the NI-RSM methanolic extract seemed to be characterized by acyl transfer reactions that reduce the overall yield of free SA release.

Conclusion

It is possible to obtain free CA from sunflower meal and free SA from rapeseed meal by alkaline hydrolysis and solvent extraction [START_REF] Vuorela | Quantitative analysis of the main phenolics in rapeseed meal and oils processed differently using enzymatic hydrolysis and HPLC[END_REF], Zago et al., 2015b), but in high temperature, strong base and toxic solvent conditions, which runs counter to a 'green' process. Here we showed that A. niger AnFaeB, ChlE and AnFaeA were very efficient catalysts for the enzymatic release of CA and SA from both industrial and non-industrial meals. To the best of our knowledge, this is the first time that AnFaeB and ChlE were applied on sunflower meals as raw natural substrates.

AnFaeB was able to hydrolyze all the mono-and di-caffeoylquinic acid isomers, together with the coumaroylquinic and feruloylquinic acid isomers identified in SFMs, whereas ChlE was unable to hydrolyze 3-CQA. The non-industrial meals processed under mild thermal conditions presented the advantage of higher initial phenolics contents. However, phenolic acid release was hampered by phenolic oxidation sidereactions, seemingly due to meal endogenous PPO-type activities, which is a major drawback for industrial applications. Here we found evidence that using an intermediary step of phenolic extraction with methanol, before enzymatic hydrolysis, could overcome this drawback for NI-SFM but not NI-RSM due to AnFaeA transesterification sidereactions. Nevertheless, this acyl transfer activity of AnFaeA could prove beneficial for the enzymatic synthesis of novel sinapic and ferulic acid esters with sugars or polyols.

Of all the substrates tested for enzymatic hydrolysis, industrial rapeseed meal and nonindustrial sunflower meal extract showed the best compromise between the three following parameters: initial TPC, hydrolysis yields, and amounts of CA and SA released. The proof of concept of such an enzymatic hydrolysis process was already successfully scaled-up in a 10-L bioreactor in the case of FaeA and raw industrial RSM [START_REF] Odinot | A two-step bioconversion process for canolol production from rapeseed meal combining an Aspergillus niger feruloyl esterase and the fungus Neolentinus lepideus[END_REF]. In addition, regarding enzymatic hydrolysis applied to meal methanolic extract, the extraction of phenolics by ethanol (less toxic and biosourced solvent, almost as efficient as methanol) followed by concentration/desolventization steps could be easily implemented at industrial scale as it is already done with hexane for instance. In the case of sunflower meal extract, this process may be advantageous since phenolic concentration as well as enzyme load would be better controlled, limiting matrix effect in the same time. This work opens new perspectives on the biotechnological applications of fungal Faes and ChlEs to other hydroxycinnamic acidcontaining raw materials, including green coffee bean wastes, co-products of rapeseed and sunflower crops (e.g. stems, straws, seed shells, sunflower capitula) as well as other oilseed plants (mustard, flax).

Figures captions

Fig. Values are mean ± SD (n=2). Values followed by same superscript letters are not significantly different, p ≤ 0.05 § The substrate was pre-incubated for 30 min in MOPS buffer before adding enzyme (t = 0 of enzyme incubation).

  NI-meals: Non-industrial meals NI-RSM: Non-industrial rapeseed meal Introduction

Figure 3 (

 3 Figure 3 (suggested location)

Figure

  Figure 5 (suggested location)

  Fig. 1. HPLC chromatogram of industrial SFM methanolic extract at 326 nm. Main identified

Fig. 2 .

 2 Fig. 2. Hydrolysis kinetics of the main CAEsters in industrial SFM, as a function of enzyme

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. Time course of 4-and 5-O-caffeoylquinic acid (4-and 5-CQA), caffeic acid (CA) and

  Fig.1

Fig. 2

 2 Fig. 2

  Fig. 3

  Fig. 5

. Plant materials 2.2.1. Preparation of the non-industrial sunflower and rapeseed meals (NI-SFM and NI-RSM)

	). ACS grade ethyl acetate (>99,5%) was purchased from Honeywell Riedel-de-Haën (Seelze, Germany) technology platform's pilot oilseed plant (Pessac, France), according to the following methods. Whole sunflower (Helianthus annuus L.) seeds (75 kg) were first cold flaked in a flaker (Damman-Croes N.V., Roeselare, Belgium) equipped with two smooth 500 mm-diameter contra-rotating cylinders spaced at 0.3 mm (capacity: 200 kg.h -1 ). Whole rapeseed (Brassica napus L.) seeds (120 kg) were first cold pressed on a MBU20 screw press (OLEXA®, Feuchy, France), fed at 75 kg.h -1 of seeds, to remove 75% of the initial oil amount. The residual oil from the sunflower or rapeseed press cakes was further extracted by steeping in hexane at 50°C, in a 480 L-capacity Guedu Pilot Agitated Filter Dryer (De Dietrich Process Systems, Semur, France). Extraction was performed on 73 kg of each press cake by immersion in hexane for 15 min followed by 2.2NI-SFM and NI-RSM were prepared at OLEAD oilseed-protein crops filtration.

Table 4

 4

(suggested location)

Table 1 .

 1 Lipid and moisture content of SFM and RSM samples # .

		Lipid content (% DM)	Moisture (% DM)
	NI-SFM	1.5 ± 0.0	7.3 ± 0.8
	I-SFM	1.9 ± 0.3	10.5 ± 0.7
	NI-RSM	1.4 ± 0.0	13.1 ± 0.3
	I-RSM	1.5 ± 0.06	10 ± 0.1

# Values are mean ± SD (n=3).

Table 2 .

 2 Phenolic content of Non-Industrial and Industrial SFM # Values are mean ± SD (n=3). Values followed by same superscript letters are not significantly different, p ≤ 0.05. * TCAD: total caffeic acid derivatives in SFM, expressed in µmol CAE/g DDM. CAE: caffeic acid equivalents. DDM: defatted dry matter.

		NI-SFM		I-SFM
		Before alkaline	After alkaline	Before alkaline	After alkaline
		hydrolysis	hydrolysis	hydrolysis	hydrolysis
	Chlorogenic acid (µmol CAE/g DDM)	55.8 ± 0.9 b	n.d.	41.4 ± 2.5 c	n.d.
	Caffeic acid (µmol/g DDM)	1.2 ± 0.0 e	n.d.	2.1 ± 0.5 d	n.d.
	TCAD* (µmol CAE/g DDM)	71.7 ± 0.5 a	n.d.	57.4 ± 1.5 b	n.d.

#

Table 3 .

 3 Phenolic content of Non-Industrial and Industrial RSM # . Values are mean ± SD (n=3). Values followed by same superscript letters are not significantly different, p ≤ 0.05. * TSAD: total sinapic acid derivatives in RSM, expressed in µmol SAE/g DDM. SAE: sinapic acid equivalents. DDM: defatted dry matter.

		NI-RSM		I-RSM
		Before alkaline	After alkaline	Before alkaline	After alkaline
		hydrolysis	hydrolysis	hydrolysis	hydrolysis
	Sinapine (µmol SAE/g DDM)	31.7 ± 0.7 c	--	21.6 ± 0.2 d	--
	Sinapic acid (µmol/g DDM)	3.9 ± 0.1 e	62.5 ± 1.8 a	2.2 ± 0.0 f	46.8 ± 3.0 b
	TSAD* (µmol SAE/g DDM)	64.2 ± 1.2 a	62.5 ± 1.8 a	44.8 ± 0.2 b	46.8 ± 3.0 b

#

Table 4 .

 4 Concentration of caffeic acid (CA), CAEsters and total caffeic acid derivatives (TCAD) before and after hydrolysis of NI-SFM methanolic extract with AnFaeB and ChlE at 50°C # . TCAD: total caffeic acid derivatives; CAE: caffeic acid equivalent. DDM: defatted dry matter. Values are mean ± SD (n=2). Values followed by same superscript letters are not significantly different, p ≤ 0.05. # 10 min incubation for AnFaeB, 60 min incubation for ChlE.§ The substrate was pre-incubated for 30 min in MOPS buffer before adding enzyme (t = 0 of enzyme incubation).

		Initial content in the meal	AnFaeB	ChlE
	4-and 5-CQA (µmol CAE/g DDM)	55.8 ± 0.9 c	--	2.0 ± 0.1 h
	3-CQA (µmol CAE/g DDM)	8.9 ± 0.4 e	--	7.8 ± 0.1 f
	3,5-di-CQA (µmol CAE/g DDM)	4.0 ± 0.2 g	--	0.6 ± 0.0 j
	4,5-di-CQA (µmol CAE/g DDM)	1.8 ± 0.1 g	--	--
	CA (µmol/g DDM)	1.2 ± 0.0 i	68.1 ± 1.7 ab	55.4 ± 0.3 cd
	TCAD* (µmol CAE/g DDM)	71.7 ± 0.5 a	68.1 ± 1.7 ab	65.8 ± 0.4 b
	CA yield (%) based on initial TCAD in meal	--	95.0	77.2
	CA yield (%) based on
	TCAD at t = 0 of enzyme	--	100.4	84.2
	incubation §

*

Table 5 .

 5 Maximal amount and yield of sinapic acid (SA) after enzymatic treatment of NI-RSM and the corresponding methanolic extract with AnFaeA.

		NI-RSM	NI-RSM
	NI-RSM	methanolic	methanolic
	55°C	extract,	extract,
		55°C	37°C

* TSAD: total sinapic acid derivatives; SAE: sinapic acid equivalent. DDM: defatted dry matter.

Acknowledgements

This work was funded by the Technical Centre for Oilseed Crops, Grain Legumes and Industrial Hemp (TERRES INOVIA, Pessac, France) and the Inter-Branch Organization for Vegetable Oils and Proteins (TERRES UNIVIA, Paris, France). This work was also funded and performed, in partnership with the SAS PIVERT, within the frame of the French Institute for the Energy Transition (Institut pour la Transition Energétique (ITE) P.I.V.E.R.T. (www.institut-pivert.com) selected as an Investment for the Future ("Investissements d'Avenir"). The French Government under the reference ANR-001-01 supported this work, as part of the Investments for the Future.

All the authors contributed to finalize the manuscript.

Competing interests:

The authors have declared no conflict of interest.

Aider, M., Barbana, C., 2011. Canola proteins: composition, extraction, functional properties, bioactivity, applications as a food ingredient and allergenicity -A industrial RSM methanolic extract at either 55°C (B) or 37°C (C). Before starting the enzymatic hydrolysis, the reaction mixture was pre-incubated for 30 min for homogenization. Initial PC: Initial phenolic concentration in non-industrial RSM (A) or in non-industrial RSM methanolic extract (B, C). Values are mean ± SD (n=2) and expressed as sinapic acid equivalents (SAE).

Values followed by same superscript letters are not significantly different, p ≤ 0.05.

Supplementary data.