Chloé Bailhache

Bruno Pontoire

Lucie Ribourg

Camille Jonchère

Delphine Queveau

Mark Irle

Ellen Thieffry

Alain Le-Bail

Patricia Le-Bail
email: patricia.le-bail@inra.fr

A study of cracks in dry cereal products

Keywords: crackers, breakage, gelatinization, starch, structure

This paper is a study of checking and breakage of dry cereal products like crackers. The products studied were produced on an industrial line; almost no cracks were observed for Plain crackers (PCs), whereas 100% of the crackers with topping (CTs) exhibited cracks. Environmental SEM images suggested that miniature cracks initiated around the piercing holes of the crackers and then extended during storage.

A study of the impact of each ingredient and of their interaction on the degree of starch gelatinization was carried out using a simplified model system (wheat flour, water and gradual enrichment until a model cracker dough was obtained). The addition of salt or sugar to flour at 40% water content shifted the starch gelatinization temperature slightly higher in comparison to a control mixture (only flour and 40% water content). A comparison of the residual gelatinization enthalpy between the PCs and the CTs made during storage (up to 3 weeks) showed that the degree of gelatinization of the CTs was more significant than the PCs (especially in the center) because of the presence of water in the topping in the center of the cracker. X-ray diffraction confirmed that the center of the CTs was indeed very different from the PCs.

Introduction

Dry cereal products (DCP) such as crackers, biscuits and cookies undergo a very specific baking process combining a baking followed by a drying until reaching a final water content in the range of 3-5 % on a dry basis (DB). DCP are generally classified by the consistency of the dough [START_REF] Whiteley | Biscuit Manufacture : Fundamentals of In-line Production[END_REF]; hard or semi-hard doughs are usually related to salted, unsweetened and semi-sweet biscuits.

This includes all types of crackers, puff pastries and semi-sweet biscuit varieties. In addition to having low sugar, or even no sugar, the fat content rarely exceeds 22% of the quantity of flour, except in the case of puff pastry [START_REF] Whiteley | Biscuit Manufacture : Fundamentals of In-line Production[END_REF]. This category of DCP differs from bread and cakes in terms of crispiness and a water content usually below 5%, providing a fairly long shelf life [START_REF] Wade | Biscuits, cookies and crackers[END_REF]. The type of cracker considered for this study are "hard dough" type crackers and more precisely in the group "savory or snack crackers" [START_REF] Manley | Manley's technology of biscuits, crackers and cookies (Tec & Nut)[END_REF].

In the cracker industry, certain groups of products are renowned for breakage at variable times after baking as a result of a hairline cracking phenomenon known as "checking". Cracker checking is usually observed as cracks which extend partway across a central portion of the biscuit rendering it very prone to complete breakage on subsequent handling [START_REF] Dunn | Factors influencing checking in biscuits[END_REF][START_REF] Saleem | Modelling biscuit checking using the finite element method[END_REF]. The mechanisms of the crack phenomenon seem to find their explanation during the cooling phase of crackers. Indeed, once the crackers exit the oven, a water gradient is present within the product with higher water content at the center than at the periphery. This moisture gradient can be attributed to baking but also to the evolution of the distribution of water during baking among the various constituents (starch, proteins etc). This gradient will drive water transfer within the cracker in order to reach equilibrium. The sections of the crackers that will uptake water will expand whereas the sections that will lose water will shrink, resulting in thermo and/or hydro mechanical stress that may cause cracks and eventually breakage, particularly if the product is in a glassy state [START_REF] Manley | Technology of Biscuits, Crackers, and Cookies[END_REF].

The use of topping, usually rich in water, increases the risk of breakage due to the setting of higher moisture content in the surface onto which the topping has been added. In addition, the presence of a higher amount of water is supposed to modify the starch gelatinization process, which is strongly dependent on the availability of water.

This work aims at elucidating the precise impact of an added topping on the risk of cracker breakage.

To address this objective, a comprehensive study based on a multi-scale approach has been considered covering microscopic and molecular scales by focusing on the interactions between the different constituents of the dough but also by relying on the role and distribution of the water during baking, post baking cooling and storage.

Materials and methods

Cracker making

The crackers considered in this study were produced in an industrial baking line. For the crackers with topping (CT), a specific topping was used as shown in Figure 1. The precise recipe of the topping cannot be revealed for confidentiality reasons. The water content of the topping added on the dough cracker top is 61.4 ± 0.6% in dry basis before baking. The addition of the topping before baking can therefore have a significant impact during baking since it causes a very substantial water intake in the center of the cracker. For this reason, analyzes were done on CTs and on PCs, in order to observe the impact of the topping on the final structure of the products.

The cracker dough consists mainly of flour, fat, sugars and salt in the proportions shown in Figure 1a.

The dough contains 38% water in dry basis. The composition of dough pieces with addition of topping was presented in Figure 1b.

The cracker with topping (CT) before baking is composed of dough and topping; representing respectively 79% and 21% of the final dough. As the topping has a water content of 61.4 ± 0.6% dry basis, the 21% of topping are decomposed into 8.1% dry topping and 12.9% water. CTs therefore contain 43% water compared to 38% for PCs, ie a 5% increase in water by adding the topping to the surface of the product.

The dough was laminated very finely and was baked in a conveyor oven. The set of steps in the preparation of crackers is very close to the classic biscuit production scheme presented by [START_REF] Manley | Biscuit, cookie and cracker manufacturing manuals[END_REF] with preparation of puff pastry, rolling using three sets of sizing rollers to reduce the thickness, the relaxation of the dough, the imprint to give the shape of the biscuit, the piercing, and the removal of a possible topping. The industrial oven used on the line of crackers is divided into 5 zones. The average baking temperature is from 200°C and the average baking time is from 4 minutes.

At the exit of the oven, the crackers were sprayed with oil, packaged and stored.

The experiments were carried out on 3 different productions and on 3-5 samples per experiment, for PCs and CTs.

Cracker storage conditions

The PC and CT crackers studied had followed the industry partner's normal post cooking procedure, i.e. they were stored in a plastic pouch within a large storage bin for 4 weeks at around 20 °C. The crackers were analyzed at the end of baking and before storage (week 0) and after storage in bins for 1, 2 and 3 weeks in order to follow the evolution of any cracks and to assess the degree of staling.

Visual observation of cracks

PC and CT were observed by environmental scanning electron microscopy (ESEM Quanta 250, FEI, Holland) used in "Lo Vac" mode. This mode is recommended for "dry" samples. The maximum chamber pressure was 130 Pa and, consequently, the samples dried slightly whilst being observed.

Sorption isotherm

The PC and CT samples were ground and placed in sorption flasks containing the following saturated saline solutions LiCl, MgCl2, Mg(NO3)2, NaCl, KCl, BaCl2 with relative humidities of 11.1%, 32.7%, 54.4%, 75.3%, 84.3%, 90.2% respectively. The flasks were set under vacuum and after a sufficient storage time (2 weeks), the water content was measured by Thermo Gravimetric Analysis. The water content of the sample was determined using the equation (1) below with S: water content, mw: wet mass and md: dry mass (Table 1).

= (

) * 100

( 1 )

Freezable water content

DSC thermograms were recorded with a micro differential scanning calorimeter MicroDSC7 evo (SETARAM instrumentation, Caluire, France). The equipment was calibrated with indium. Stainless steel pans were used and an empty pan was taken as a reference.

PCs and CTs were studied before and after equilibration in the flasks containing saturated saline solutions. Samples were collected and placed on the pan and the pan was hermetically closed. The DSC scans were performed from 20 to -20 °C ; -20 to -30°C and from -30 °C to 20 °C at a scanning rate of 0.3 °C/min. A 10 minutes plateau was imposed between each temperature ramp.

Measurement of lipid content in a cracker

Lipid content in PCs and CTs was measured with the Bligh and Dyer method, which is a cold lipid extraction method. The proportions of chloroform / methanol / aqueous phase were 2/2/1.8 v/v/v. A biphasic system was obtained: methanol and water constitute the upper phase and the chloroform phase, which contains lipids, corresponds to the lower phase. 50 mL capped glass tubes were used, in which about 1 g of sample, 10 mL of methanol, 10 mL of chloroform and 5 mL of 150 mM NaCl were placed. Tubes are vortexed for one minute, decanted for 30 s, and then centrifuged for 15 min at 2000 rpm at 15°C (JOUAN GR2022 rotor 99mm centrifuge). The lower lipid-containing phase was recovered, filtered through a funnel containing glass wool and anhydrous sodium sulfate and collected in a previously weighed glass flask (m1). The upper phase was washed twice more with 10 mL of chloroform, vortexed again, centrifuged and the lower phase recovered. Then the solvents were evaporated with a rotary evaporator (Büchi Rotavapor R-200) with a water bath at 30°C (Büchi Heating Bath B-490). The last traces of solvent were removed under a stream of nitrogen (N-EVAP 111 Nitrogen evaporator). The balloon was again weighed (m2). The mass remaining in the flask determines the amount of lipids extracted m = m2-m1. Lipid content in crackers was expressed in g / g dry crackers, then in g / g dry flour. The same method was used for the dough which was first freeze dried to reduce its water content (Lyolab 3000 freezedryer).

Glass temperature determination

DSC thermograms were recorded with a differential scanning calorimeter DSC Q100 (TA Instruments, Guyancourt, France). For the analysis of the glass transition temperature (Tg), CTs and PCs were taken directly from the factory after their manufacture. Five crackers of each type are recovered after 7 hours of storage and crushed. 50mg of ground material is introduced into the cell, the reference cell is empty. The kinetics chosen for this study is as follows: a first temperature decrease from 20 to -80 ° C to 10 ° C / min ; a temperature rise from -80 to 180 ° C to 10 ° C / min and a second temperature drop from 180 to 20 ° C to 10 ° C / min. The determination of the glass transition temperature is made during the rise in temperature.

Assessment of starch gelatinization by thermal analysis

DSC thermograms were recorded with a differential scanning calorimeter DSC Q100 (TA Instruments, Guyancourt, France). The assessment of non-gelatinized starch was determined as follows: 20 mg of sample were placed on to the DSC pan and 30 mg of water were added in order to run a temperature scan in excess of water to ensure a full gelatinization. Water was previously stirred for 10 min with nitrogen bubbling to prevent oxidation. Each run was made of a temperature scan at 3 °C/min from 10 to 160 °C. A final temperature of 120 °C was in fact sufficient to detect the gelatinization peak.

DSC tests were done in triplicate and the thermograms were analyzed (gelatinization peak) to determine the gelatinization enthalpy in Joule/g of dry flour.

Assessment of starch retrogradation and starch complexation by X ray diffraction

The edge and center of the crackers are separated using a cookie cutter after a specific storage time.

About 50 mg of each sample was sealed in a copper ring between two adhesive tape sheets to prevent any change in water content and examined by Wide Angle X-ray Scattering. Measurements were performed using a D8 Discover spectrometer from Bruker-AXS. Cu Kα1 radiation (Cu Kα1 = 1.5405 Å), produced in a sealed tube at 40 kV and 40 mA, was selected and parallelized using a double Gobël mirror parallel optics system and collimated to produce a 500-µm beam diameter with sample alignment by microscopic video and laser. Data were monitored by a GADDS 2D detector for 10 minutes and normalized.

Model systems preparation

Model systems were prepared by hand mixing with a spatula for 5 min. The four principal ingredients (flour, salt, sugar and fat) were always in the same proportions which were similar to those of the real dough. The model dough was built and evaluated progressively. The impact of each ingredient and the interactions between ingredients were studied by making doughs with either two, three or four ingredients; the last one represents a complete model system with all ingredients (final model system). The different model systems were prepared with 40% water content (dry basis) and represented by the following formula (2) where F, S, Su, FA and W refer to flour, salt, sugar, fat and water respectively with a,b,c,d representing mass ratio that were calculated on a dry matter basis (a+b+c+d=100). The y mass ratio represents the total water content, which was always measured on a humid basis). The ingredients were added while respecting the recipe of the reference crackers.

(FaSbSucFad)xWy

( 1 )

Statistical analysis

Statistical analysis was performed on the basis of results obtained at least in triplicate and on three different productions. The average and standard deviation were calculated for each experiment.

Results and discussion

The fragility of dry products like crackers causes the appearance of cracks (rift) that may result in cracker breakage (dislocation of the cracker in 2 or several parts). A test has been established to count and locate the cracked or broken crackers.

Counting cracks on PCs and CTs during storage

The counting of cracks was performed on 100 PCs and 100 CTs at T0+8H (8 hours in storage bin) by visual observation. No crack was detected on all the examined PCs (Figure 2a). On the other hand, the observation of CTs reveals a crack rate of 67% with cracks mostly visible on the bottom surface (Figure 2b), but also present on the top of the crackers, that is to say on the topping. Then, for the 33% showing no crack, a second analysis was performed which consisted of carefully scratching the topping with a scalpel to look at the top surface of the crackers under the topping. These observations revealed that all the remaining crackers had cracks under the topping, which were not visible to the naked eye before removing the topping. Thus all the samples observed with topping already all have cracks eight hours after their production. These products (CTs) are therefore likely to be much more fragile and sensitive to manipulation, unlike PCs that had no cracks.

SEM Observation

PCs and CTs were observed in an environmental scanning electron microscopy (ESEM). Many blisters were visible on the surface of the PCs but no cracks were observed on either side of the PCs.

Whereas for the CTs, cracks were visible on both sides of the crackers. A zoom of the termination of the crack was made with x600 magnification (Figure 2c). This refined observation showed that the cracks started from the piercings of the dough before baking. The image reveals a sort of extension of the crack, and these "ways" containing fewer starch granules on the bottom surface of several CTs continue for a long distance, sometimes even to the edge of the cracker. These seem to be at the origin of crack extension and finally of cracker breakage. A mechanical shock may also contribute to provoke breakage but it clearly appears that these weaker domains (path containing less starch granules) were more often observed in CTs (Figure 2c).

Indeed, most of the cracks observed on the CTs were passing through one or several piercings. The weaker domains observed on the CTs may be responsible for the fragility of the cracker during cooling (Figure 2d). However, these cracks as well as these crack paths were not present on the PCs.

Connection between the piercing points did not seem to be the only explanation of cracks in CTs.

Calculation of the amount of freezable water

The total water content was evaluated by Thermo Gravimetric Analysis 24 hours after the cooking.

The values for PCs and CTs were respectively 2.7 ± 0.2; and 4.5 ± 0.5 (g water/g sample) corresponding to 0.042 ± 0.003 and 0.084 ± 0.009 (g water/g dry flour). This shows that the topping significantly increases the water content for the CTs.

The amounts of free or bound water were expressed in grams per gram of dry flour. Indeed, the mass of dry flour used is identical in a cracker before and after baking and with or without topping.

This makes it possible to compare the values obtained between the two types of crackers.

After the sorption equilibria in the different saturated saline solutions, the total water content of PCs and CTs (g water/g dry flour) was obtained and presented by the sorption isotherms in Figure 3.

Although the general appearance of the sorption curves is identical for both types of crackers, there is a clear difference between PCs and CTs; the presence of topping seems to lead to greater water absorption. This phenomenon seems to increase with increasing water activity.

The amount of freezable water was determined by calorimeter (Le-Bail, Chapleau, Anton-De Lamballerie, & Vignolle, 2008). Figure 4 shows DSC heating curves of PCs and CTs after sorption equilibria and cooling from 20 to -30°C at a scanning rate of 0.3°C/min. On the heating thermogramm, a double endothermic transition was observed between -20°C and -4 °C; it was attributed not only to the ice melting [START_REF] Pradipasena | Temperature Dependence of Fraction of Frozen Water in Solutions of Glucose and its Oligomers, Dextrans, and Potato Starch[END_REF][START_REF] Tran | Effect of starch modifications and hydrocolloids on freezable water in cassava starch systems[END_REF] but also to the lipid phase and to the topping for the CTs (verified by DSC of the lipids -not shown).

The integration of the double peak indicates the amount of heat Q1 (J/gsample) needed to melt the ice contained in the product. By dividing this amount by the latent heat of pure water (333.3 J/g) one can obtain the amount of water frozen per gram of sample. The unfreezable water can be calculated from the difference between the total water of the sample and the freezable water. However, in this particular case, it is necessary to determine the amount of lipids present in the crackers at the end of the process in order to subtract the thermal effect of the lipid during the DSC runs. Lipid content in crackers and in the dough was measured with Bligh and Dyer method and the amount of the freezable lipid phase of the both crackers was defined by calorimeter.

The quantities of lipids and water in the free and bound states were expressed as before in grams per gram of dry flour. Figure 5 shows the amount of bound water in grams of water per gram of dry meal in the PCs and CTs on the day of production and after equilibration over saturated salt solutions.

At D0, the PCs have 100% bound water (0.042g bound water / g dry flour) whereas the CTs have more (0.075g bound water / g dry flour) and possess a small quantity of free water (0.01g free water / g dry flour), which can be attributed to the topping. During conditioning in relative humidities created by the saturated salt solutions the crackers absorbed water. The evolution of the bound water for the different salt solutions is identical for both types of crackers but the amount of bound water was always greater for crackers with topping and increases with the ERH% (Water activity).

The detection of the amount of free water was observed starting at different level of water activity for the CTs and the PCs. In the case of CTs, 2% of the water was observed as being free at a relative humidity of 75.3%. At 90.2% relative humidity, 18% of the water was present as free water for CTs, while the PCs only had 5% free water present. The free water detected in the CTs corresponds to the water absorbed in a big part by the topping.

Interactions between dough ingredients in the presence of water

This part of the study aims to understand the effect of the interactions between the ingredients on the phase changes occurring in the cracker dough (38% water content dry basis) during baking. To do this, hydrothermal transitions were studied using a simplified model dough made of wheat flour, water and of other ingredients whose proportions were progressively increased until reaching the reference "model dough" recipe. This reference model dough was then compared to the "real" cracker dough.

A preliminary investigation was done to determine the impact of the ingredients on the hydrothermal transitions of starch. The impact of the water content on the evolution of the gelatinization profile of the flour was carried out at different water contents (30%, 40%, 50%, 60%, and 80% on dry basis) by DSC.

In the literature, many authors have shown that the starch gelatinization temperature in wheat flour dough is influenced by the water content as well as by the other wheat flour constituents, such as proteins [START_REF] Ghiasi | Effects of flour components and dough ingredients on starch gelatinization[END_REF][START_REF] Zobel | Starch Gelatinization: An X-ray Diffraction Study[END_REF][START_REF] Rolée | Effect of Moisture Content on the Thermomechanical Behavior of Concentrated Waxy Cornstarch -Water Preparations -A Comparison with Wheat Starch[END_REF][START_REF] Liu | Phase transition in potato starch -water system I . Starch gelatinization at high moisture level[END_REF][START_REF] Mohamed | The effect of mixing and wheat protein / gluten on the gelatinization of wheat starch[END_REF]. The comparison of thermograms of F as a function of the water content (Figure 6a) shows that in excess of water, the sample (F100S0Su0Fa0)17W83 had a single endotherm "Ge". This endotherm corresponds to the phenomenon of gelatinization of the starch [START_REF] Donovan | Phase transitions of the starch-water system[END_REF]. The gelatinization temperature corresponding to the bottom of the endotherm (TGe) observed for these samples was 60.5°C ± 0.5.

The lipid fraction of the flour F being about 1%, the second endotherm M2 characteristic of the fusion of complexes formed by amylose during gelatinization with the endogenous lipids of flour is clearly visible TM2 = 92°C ± 0.4 [START_REF] Biliaderis | Thermal Behavior of Amylose-Lipid Complexes[END_REF]. As the amount of water decreases, the first endotherm Ge has a shoulder for the higher temperatures. This endotherm then transforms into an endotherm M1 corresponding to the fusion of the starch, which will move towards higher temperatures with decreasing water content. In parallel, the endotherm Ge decreases and eventually disappears. The M2 fusion endotherm of complexes was also shifted to higher temperatures, but in a more progressive way; for the lowest levels of water content, the M2 endotherm tended to merge with the M1 endotherm.

The interactions between the different ingredients of the dough were first studied in model systems composed of two ingredients. These model systems provided an indication of the effects of fat, salt and of sugar on starch gelatinization. The recipes of these systems were calculated from the final batter model systems respecting the mass ratio between the ingredients.

In general, the binary systems thermograms (Figure 6b) appear similar to flour system (F100S0Su0Fa0)60W40. The fat did not affect starch gelatinization at a 40% water content, in agreement with previous studies such as [START_REF] Eliasson | Interactions between starch and lipids studied by DSC[END_REF] and [START_REF] Chiotelli | Effect of triglycerides on gelatinisation and rheological properties of concentrated potato starch preparations[END_REF]. On the other hand, the addition of salt or sugar to flour at 40% water content ((F99S1Su0Fa0)60W40, (F92S0Su8Fa0)59.5W40.5 systems) tends to shift the starch gelatinization temperature towards a higher temperature (69°C and 70°C respectively) in comparison with the mixture (F100S0Su0Fa0)59W41 (61°C) (Table 2). This shift in the gelatinization endotherm is related to the presence of salt or sugar, which competes with starch for water; in turn, less water is available for the gelatinization of the starch. Indeed, the increase in the gelatinization temperature has been attributed to the solute's ability to limit the availability of water for starch, i.e., to reduce the chemical potential of water [START_REF] D'appolonia | Effect of bread ingredients on starch gelatinization properties as measured by the Amylograph[END_REF][START_REF] Spies | Effect of sugars on starch gelatinization[END_REF]. However, the effect of sucrose and salt on starch gelatinization was not solely due to the decreased availability of water, because the gelatinization energy did not decrease progressively and at the same rate as the amount of sucrose or salt in aqueous phase [START_REF] Wootton | Application of Differential Scanning Calorimetry to Starch Gelatinization. I. Commercial Native and Modified Starches[END_REF].

Consequently, more complex systems (Figure 6c) (ternary and final model system) were prepared, to understand the behavior of the real system. The different endotherms that were obtained can be explained in the same way as the final model system (Figure 6d). A difference between the two systems was found; it could be explained by the difference in the preparation and mixing method between the model system and the real batter. In model systems, the mixing was done manually while in the real batter (real cracker recipe) was mixed in an industrial way. In addition, it can be also attributed to the decrease in the water content, which was 41% and 38% (dry basis) for the final model system and the real batter, respectively.

Nevertheless, this approach explains that the interactions between the dough ingredients, the water limitation and probably the mode of preparation of the dough are the major factors responsible for the shift and combination of the different phase transitions observed during baking.

This study allowed us to locate on the phase diagram the melting temperatures Tm = (TGe + TM1) / 2 of the biscuit flour used in this study compared to the Tm of wheat starch (Figure 7). It is known in the literature that the thermograms obtained by DSC for wheat flour are identical in appearance to those obtained for wheat starch with the same water content. Indeed, [START_REF] Hoseney | Wheat gluten -A glassy polymer[END_REF] showed that wheat gluten hydrated at 60% did not show a peak of protein denaturation or other;

which testifies to the lack of long-distance order in the gluten molecule. [START_REF] Eliasson | Thermal stability of wheat gluten[END_REF] were able to identify transitions that could be attributed to gluten proteins, but the enthalpies were less than 0.1 cal / g gluten. It can therefore be considered that the thermal denaturation of gluten is not detectable in DSC. The thermograms of wheat flour can therefore be interpreted as representing the thermal behavior of starch alone. The gelatinization temperature is slightly higher for flour which may be due to competition for water between starch and gluten. Eliasson in 1983 assumes that the diffusion of water into the starch granules can be delayed by the presence of gluten on their surface.

This study has shown that the model systems composed of four ingredients (F, S, Su, Fa) make it possible to explain the different phase transitions in the real system but the temperatures obtained are strongly shifted. The model systems approach explained that the interactions between dough ingredients, water limitation and probably the dough preparation mode are the main factors responsible for the shift and the combination of the different phase transitions observed during baking.

Assessment of starch gelatinization in the crackers

A sample was taken from a crushed cracker; A DSC run was then carried out with this sample in excess of water (60% according to the protocol previously described in materials and methods). The thermograms obtained for the CTs and PCs are presented in Figure 8. They show the presence of a gelatinization endotherm (G), which indicates that the starch present in the sample had been only partially gelatinized during the baking process. In addition, a second endotherm was observed around 100 °C; such endotherm is characteristic of the fusion of amylose/lipid complexes (M2). The residual gelatinization temperature was 66 °C ±0.3 °C and was the same for both PCs and CTs samples.

The residual gelatinization enthalpies were ∆HR =3.2 ± 0.1 (J/g of dry flour) and 5.1 ± 0.5 (J/g of dry flour) for CTs and PCs samples respectively. The enthalpy of the cracker dough in excess of water was also determined by DSC as being ∆HT =8.2 ± 0.5 (J/g of dry flour). ∆HT represents the total gelatinization enthalpy; in excess water conditions the starch has sufficient water to swell and undergo a complete leaching of the amylose and amylopectin macromolecules out of the granule.

The enthalpy of partial gelatinization ∆HP is therefore ∆HT-∆HR J/g of dry flour: Values of ∆HP=5.0 ± 0.5 (J/g of dry flour) and ∆HP=3.1 ± 0.5 (J/g of dry flour) were observed for the CTs and the PCs respectively corresponding to 61% and 38% of gelatinized starch for CTs and PCs respectively. The presence of the topping in CTs provides extra water to the cracker during baking and allows a higher ratio of gelatinization in the center of the cracker. A new study was then conducted by taking a sample at the center of the cracker and another at the edge to determine the partial enthalpies in the periphery and in the center of the CTs. For CTs, the partial gelatinization enthalpies in the periphery and in the center were respectively ∆HPP= 4.4 ± 0.3 (J/g of dry flour); ∆HPC= 5.5 ± 0.5 (J/g of dry flour).

The gelatinization of CTs was therefore more significant than for PTs; this is attributed to the presence of water provided by the addition of topping in the center of the cracker. In fact, the gelatinization of the starch during the baking of CTs is not done in a homogeneous way in the cracker, the water brought by the topping benefits essentially to the dough located directly underneath.

Assessment of starch retrogradation and starch complexation

PCs and CTs were stored in closed bins at room temperature. Samples were taken from both types of crackers at their edges and the centers after 7 h, 24 h, 48 h and 1 week of storage, and studied by Xray diffraction as described above. The X-ray diffraction spectra of PCs (center and edge) and CTs (center and edge) are shown in Figure 9a and Figure 9b ; and Figure 9c and Figure 9d respectively as well as the spectrum of the cracker dough.

The cracker dough displayed the typical A-type crystallinity patterns (2Θ =9.7°, 11.1°, 15°, 14.9°, 17°, 18°, 19.5°, 23°, λCuKα1=0.15405nm), very frequently encountered in cereals [START_REF] Katz | Uber die Umwandlung von Stärke präparaten mit Kartoffelstärke Spektrum (B-Spektrum) in Solche mit Weizenstärke-Spektrum (A-Spektrum) und Umgekehrt[END_REF].

The spectra of the PC center and edge were identical and did not vary during the storage. They showed amorphization of the type A structure after the cracker baking, with the appearance of a diffusion bump centered on 19° in 2Θ. The gelatinization of the wheat starch, responsible for the Apolymorphic structure, is incomplete, leading to a partial destructuration of the starch granules.

The spectra of the edge of the CTs were identical to those obtained from the PCs, however, those from the center of the cracker were different. The CT center is unstructured, however, by comparing the spectra of samples PC center to the CT center, an onset of crystallization was observed with the appearance of a peak around 20° (2Θ). A V-type polymorphic structure seems to appear, masked by the large diffusion bump. This suggests that amylose reacts with endogenous lipids or exogenous fats during baking and the crystallization of amylose lipids complexes increases during the cooling and the storage period, despite the presence of all the cracker components.

The X-ray diagrams of the PCs and the CTs samples did not display the typical peaks of B-type crystallinity patterns due to the presence of amylose and/or amylopectin having retrograded during storage [START_REF] Putaux | Network formation in dilute amylose and amylopectin studied by TEM[END_REF].

A sufficient amount of water seems to be present in the dough for CT resulting in a higher rate of gelatinization of the starch in the heart of the cracker with topping and also allow the complexation of the fat with the amylose outside of the granule. On the other hand, the mobility of amylopectin and / or amylose macromolecules does not seem sufficient to allow their reorganization into double helices and to form a B type structure during storage for the two types of crackers studied.

Conclusion

This study investigated the origin of cracker breakage in the case of crackers with and without topping. As a result, it clearly appears that the topping, which is rich in water, has an impact on the degree of starch gelatinization of the dough located underneath the topping. In the case of the control cracker without topping, no major difference was observed between the center and the edge of the cracker.

A comprehensive study done on a model recipe showed that the main ingredients that were restricting the gelatinization of starch were the salt and the sugar, plus of course the water content.

The water provided by the topping resulted in a cracker with a highly heterogeneous structure, with a matrix poorly gelatinized on edge and highly gelatinized on center. Additional observation of the crackers under SEM showed that crackers with topping contained crack primers starting from the piercing done on the cracker dough before baking. Counting of crackers stored in realistic conditions showed that 100% of crackers with topping were either exhibiting cracks or were broken, whereas crackers without topping did not exhibit any cracks or crack primers. Solutions to mitigate breakage of crackers with topping could be to reduce the water content of the topping and to apply an additional thermal treatment after baking in order to reduce the water gradient inside the crackers with topping.

Acknowledgements

  This project was supported by the Région Pays de la Loire (BRICE Project), by FRANDEX (CIFRE agreement n°2014/1277), by INRA-BIA and by ONIRIS-GEPEA.

Figure 1 :

 1 Figure 1 : CTs (a) and PCs (b) composition in dry mass

Figure 4 :Figure 5 :

 45 Figure 4 : Endothermic transitions before storage (Day 0) for PCs (a) and CTs (b)

Figure 6 :Figure 7 :

 67 Figure 6 : Thermograms of flour (F) with different water content (a) ; Binary systems thermograms with 40% water content (b) ; Ternary and final model systems thermograms with 40% water content (c) ; Real cracker dough (d)

Table 1 :

 1 The water contents (g water/g dry flour) of the PC and CT samples after equilibration in the flasks

	Satured saline solutions (20°C)	PCs	CTs
	LiCl	(aw = 0.114)	0.064 ± 0.001	0.085 ± 0.004
	MgCl2	(aw = 0.313)	0.0973 ± 0.0002	0.121 ± 0.002
	Mg(NO3)2 (aw = 0.545)	0.138 ± 0.002	0.179 ± 0.008
	NaCl	(aw = 0.754)	0.235 ± 0.005	0.29 ± 0.01
	KCl	(aw = 0.851)	0.3319 ± 0.0006	0.429 ± 0.004
	BaCl2	(aw = 0.907)	0.431 ± 0.007	0.564 ± 0.005

Table 2 :

 2 Gelatinization (Ge) and fusion of the starch (M1) temperatures

	Systems	T°(C) Ge	T°(C) M1
	(F100S0Su0Fa0)17W83	60.9
	(F100S0Su0Fa0)43W57	61.0	74.3
	(F100S0Su0Fa0)51W49	61.0	79.8
	(F100S0Su0Fa0)59W41	60.8	91.1
	(F100S0Su0Fa0)68W32	60.9	110.7
	Final model system	70.6	88.7
	Real dough	92.7	111.7


Antony Van Der Ent

Ana Ocenar

Romane Tisserand

John B Sugau

Guillaume Echevarria

Peter D Erskine

Herbarium X-ray Fluorescence screening for nickel, cobalt and manganese hyperaccumulator plants in the flora of Sabah (Malaysia, Borneo Island)

Keywords: hyperaccumulators, phylogenetic, ultramafic, Phyllanthaceae, Malpighiales

Sabah (Malaysia) on the Island of Borneo has high plant diversity (>8000 species) occurring on a wide range of soils, including ultramafic soils which are known to host hyperaccumulator plants. In this study a new approach ("Herbarium X-Ray Fluorescence Ionomics") was used to obtain elemental data from herbarium specimens using non-destructive X-Ray Fluorescence spectroscopy analysis. In total ~7300 specimens were thus analysed for nickel, cobalt, and manganese concentrations at the Herbarium of the Forest Research Centre (FRC) in Sepilok, Sabah. The measurements led to recording a total of 759 specimens (originating from 17 families in 30 genera and 74 species) as trace element hyperaccumulators, including 28 nickel hyperaccumulator species (in 10 families, 17 genera), 12 cobalt hyperaccumulator species (in 3 families, 7 genera), and 51 manganese hyperaccumulator species (in 12 families, 24 genera). The outcomes of this research demonstrate that handheld XRF is highly useful approach for hyperaccumulator plant discovery in herbarium collections that has the potential to add vast numbers of hyperaccumulating taxa to the global inventory.

INTRODUCTION

Some rare plants restricted to ultramafic soils have evolved ecophysiological mechanisms to accumulate trace elements in extreme concentrations and are known as hyperaccumulator plants [START_REF] Jaffré | Sebertia acuminata: A Hyperaccumulator of Nickel from New Caledonia[END_REF][START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF]van der Ent et al. 2013a). Nominal threshold values for 'hyperaccumulation status' have been established for various elements, including 1000 µg g -1 for Ni, 300 µg g -1 for Co, and 10 000 µg g -1 for Mn [START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF]van der Ent et al. 2013a).

Hyperaccumulators can be either 'facultative' or 'obligate' hyperaccumulator plants [START_REF] Pollard | Facultative hyperaccumulation of heavy metals and metalloids[END_REF]. Obligate hyperaccumulating taxa are exclusively found on ultramafic soils where they consistently hyperaccumulate, whereas facultative hyperaccumulator taxa can also grow on nonultramafic soils where they do not hyperaccumulate [START_REF] Pollard | Facultative hyperaccumulation of heavy metals and metalloids[END_REF]van der Ent et al. 2013a).

To date more than 700 hyperaccumulator species have been reported globally of which ~ 70% are Ni hyperaccumulators [START_REF] Reeves | A global database for hyperaccumulator plants of metal and metalloid trace elements[END_REF]. Hyperaccumulators with >1 w% (i.e. 10 000 µg g -1 Ni) are also called 'hypernickelophores' and approximately 50 such taxa are presently known [START_REF] Jaffre | Accumulation du nickel par une Rubiace é de Nouvelle Cale donie, Psychotria douarrei (G. Beauvisage) Däniker[END_REF][START_REF] Reeves | A global database for hyperaccumulator plants of metal and metalloid trace elements[END_REF]. Global hotspots for hyperaccumulator plants include Brazil, Cuba, and New Caledonia [START_REF] Reeves | Nickel hyperaccumulation in the serpentine flora of Cuba[END_REF]2007;[START_REF] Isnard | How did the ultramafic soils shape the flora of the New Caledonian hotspot?[END_REF].

North Borneo (principally the Malaysian state of Sabah) is one of the top five biodiversity centres in the world [START_REF] Barthlott | Geographic patterns of vascular plant diversity at continental to global scales[END_REF]) and the influence of ultramafic soils on biodiversity in this region have been studied extensively in recent years (van der Ent et al., 2015b(van der Ent et al., , 2018)). Plant adaptation to ultramafic soils represents a valuable experiment system for evolutionary ecologists and geneticists [START_REF] Brady | Evolutionary ecology of plant adaptation to serpentine soils[END_REF]. Ultramafic soils contain toxic concentrations of some elements (e.g. Mg and Ni) and a lack of mineral nutrients (e.g. P and K), resulting in edaphic stresses for plants [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF][START_REF] Galey | Ultramafic geoecology of South and Southeast Asia[END_REF]. Ultramafic outcrops are relatively widespread and cover 4.6 % (3500 km 2 ) of the total land area of Sabah [START_REF] Repin | Serpentine ecology in Sabah, Malaysia[END_REF]van der Ent et al., 2013bvan der Ent et al., , 2015b)). In Sabah, 33 plant species are currently known as Ni hyperaccumulators, seven species as Co hyperaccumulators, and 14 species as Mn hyperaccumulators (van der Ent et al., 2015;2016;2018d;Nkrumah et al., 2018).

Globally the discovery of hyperaccumulator plants has been hindered by the lack of systematic screening of plant species and is highly biased towards particular regions [START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF]van der Ent et al. 2013a). The greatest numbers of Ni hyperaccumulator plants are from Malpighiales order [START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF][START_REF] Reeves | A global database for hyperaccumulator plants of metal and metalloid trace elements[END_REF]. The Malpighiales is one the most diverse groups of flowering plants, comprising about 8% of all eudicots and 6% of all angiosperms and has approximately 40 families containing over 16 000 species (Davis et al., 2005;[START_REF] Korotkova | Phylogeny of the eudicot order Malpighiales: Analysis of a recalcitrant clade with sequences of the petd group II intron[END_REF][START_REF] Wurdack | Malpighiales phylogenetics: Gaining ground on one of the most recalcitrant clades in the angiosperm tree of life[END_REF]. The Malpighiales has its greatest diversity in the Neo-and Old-World Tropics (Berry et al., The value of herbaria as references for taxonomic and biogeographical information on floras is widely established, but herbaria may also supply information on the elemental profile in stored specimens (van der Ent and al., 2016;Nkrumah and al., 2018a). This is not a new concept, and the use of herbarium specimens for discovering hyperaccumulator plants goes back to the 1970s, and tropical hyperaccumulators plants were studied first by Brooks and co-workers from the mid-1970s [START_REF] Brooks | Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants[END_REF]Jaffré et al., 1979;[START_REF] Kersten | Nickel uptake by New Caledonian species of Phyllanthus[END_REF]. Until the 1980's, testing for hyperaccumulation used mainly atomic absorption spectrophotometry (AAS), which can only analyse one element at a time, which limited discovery of the range of possible hyperaccumulated elements (i.e. frequently only Ni was analysed). Nowadays, trace elements in plant samples can be analysed using techniques that capture quantitative data from a suite of different elements simultaneously, such as inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively-coupled plasma mass spectrometry (ICP-MS). For example, fragments of herbarium specimens have been analysed with ICP-AES for suites of elements in the flora of the DR Congo [START_REF] Yang | Elemental levels and relationships in the Flacourtiaceae of New Caledonia and their significance for the evaluation of the 'serpentine problem[END_REF], selected families of plants in New Caledonia, and more recently in search of Mn hyperaccumulators in selected lineages in Australia [START_REF] Fernando | Foliar Mn accumulation in eastern Australian herbarium specimens: prospecting for 'new' Mn hyperaccumulators and potential applications in taxonomy[END_REF]. However, as small fragments (typically 20-30 mg) of herbarium material needs to be excised for the analysis, this is a destructive method of analysis, which is ultimately not compatible with preserving herbarium specimens for prosperity. Moreover, plant material acid digestion (or ashing) followed by ICP-AES analysis is relatively expensive and timeconsuming. In contrast, X-ray fluorescence spectrometry (XRF) is a non-destructive technique to quantitatively determine multi-elemental concentrations within a sample. Handheld XRF devices have in-built X-ray source (miniature tube) that produces an incident beam that causes a sample to emit a spectrum of fluorescent X-rays which can be interrogated to determine the concentrations of elements present. In order to obtain reliable quantitative data, the XRF instrument has to be appropriately calibrated [START_REF] Markowicz | Quantification and correction procedures[END_REF], which is particularly important for non-standard matrix samples such as herbarium material [START_REF] Gei | Tools for the Discovery of Hyperaccumulator Plant Species and Understanding Their Ecophysiology in[END_REF]. The non-destructive nature and short duration of the measurements (e.g. 30 seconds), makes it possible to perform elemental analysis on a vast number of stored herbarium specimens [START_REF] Gei | Tools for the Discovery of Hyperaccumulator Plant Species and Understanding Their Ecophysiology in[END_REF]. This approach has been termed "Herbarium X-ray Fluorescence Ionomics" and enables to obtain elemental information from entire phylogenetic lineages, and as such is expected to be transformative for the discovery of hyperaccumulator plants (van der Ent et al., 2019).

The flora of Sabah was already known to be rich in hyperaccumulator plant species (van der Ent et al., 2015;2016;2018d), but these discoveries originated from field-collected material during ecological investigations. It was envisaged that the flora of Sabah would harbour numerous more hyperaccumulator species hitherto unknown, that could be discovered through analysis of herbarium specimens. Therefore, in this study ~7300 herbarium specimens were measured with handheld XRF for Ni, Co and Mn concentrations at the Forest Research Centre (FRC) Herbarium at Sepilok in Sabah, Malaysia. The aim was to prospect for novel hyperaccumulator plants to contribute to a broader understanding of the phylogenetic incidence of hyperaccumulation, and to allow for the identification of species potentially suitable for utilization in phyto-technologies, such as agromining (e.g. the use of hyperaccumulator plants to obtain Ni from ultramafic soils).

MATERIALS AND METHODS

Selection of herbarium specimens for the XRF scanning

Given that the FRC Herbarium contains ~350 000 specimens (~8000 species or 10 000 taxa) from the state of Sabah, a selection had to be made. All specimens in the Malpighiales families Phyllanthaceae, Salicaceae and Violaceae (totalling 5592 specimens) were selected. The selection further contained the genus Walsura (Meliaceae -98 specimens), the genus Mischocarpus (Sapindaceae -81 specimens), and the family Sapotaceae (all genera -383 specimens). This selection of specimens was based on previous studies on hyperaccumulator plants in Sabah (van der Ent et al., 2015;2016;2018d). Furthermore, single specimens from all species (1183 specimens) known to occur on ultramafic soils in Sabah were selected to provide a wide phylogenetic assay of the incidence of hyperaccumulation. In total, this resulted in 7337 specimens that were measured.

XRF calibration for measurement of herbarium specimens

The Thermo Fisher Scientific Niton XL3t 950 GOLDD+ analyser uses a miniaturised X-ray tube (Ag anode, 6-50 kV, 0-200 µA max) as its excitation source. The X-ray tube irradiates the sample with high-energy X-rays which excite (characteristic) fluorescent X-rays in the sample. These fluorescent X-rays are detected and quantified with a large 20 mm 2 Silicon Drift Detector (SDD, 185 eV, up to 60 000 counts per second). It can detect elements from Mg to U within 15-60 seconds with absolute detection limits of 50-100 µg g -1 (but typically >300 μg g -1 depending on the element in real-life samples). The instrument incorporates Compton Normalisation; appropriate for the relatively low elemental concentrations found in plant material in a low-density (i.e. cellulose) matrix.

A total of 590 dried plant samples were used from earlier ecological studies in Sabah (van der Ent et al., 2015b;van der Ent et al., 2016b). These plant samples covered concentration ranges ranging from 'normal' to abnormal (i.e. hyperaccumulation) and includes known Ni, Co and Mn hyperaccumulator samples. From each sample, 6 mm diameter leaf discs were extracted using a paper punch. The XRF analysis was undertaken on a sheet of 'herbarium cardboard' on a pure titanium plate (~99.995%, 2 mm thick × 10 × 10 cm) to provide a uniform background and block transmitted X-rays. The XRF analysis used the 'Soils Mode' in the 'Main filter' configuration for 60s duration. After scanning, the leaf samples were weighed and pre-digested in 1 mL 70% HNO3. The samples were subsequently digested at 125°C for 2 hours and diluted to 30 mL with ultra-pure water before analysis with ICP-AES (Varian Visa Pro II), which included measurements of the following elements: Ni, Co, Cr, Cu, Zn, Mn, Fe, Mg, Ca. The quality control included the NIST 1515 ('Apple Leaves') standard and blanks.

Herbarium specimen and sample contamination

Each herbarium specimen was measured for 30 seconds in 'Soil Mode' with the 'Main filter' and the raw XRF data was corrected using the regression formulas obtained from the empirical, calibration.

The pure titanium plate was located under the herbarium specimens to provide a uniform background and to block penetrating X-rays. Consistently, old/mature leaves (i.e. the most basal leaves on a specimen) were measured, and care was taken to fully cover the 6 mm measurement area of the XRF.

The potential for contamination of leaves with soil particulates presents a problem for the accurate determination of elemental concentrations in plant material [START_REF] Gei | A systematic assessment of the occurrence of trace element hyperaccumulation in the flora of New Caledonia[END_REF]. This risk is highest for ground-herbs, and lesser for tall trees, but cannot be entirely avoided. Concomitant apparent high foliar concentrations of Cr (>60 μg g -1 ) and Fe (>2500 μg g -1 ) are useful indicators for soil contamination, as these elements are major constituents of ultramafic soils. Any suspect samples with anomalously high Cr and Fe were excluded from the dataset. Another possible problem is the practise of some tropical collectors to use methylated spirit for temporarily preserving sample collections to limit decomposition of the specimens during transport, which can potentially 'wash out' soluble elements from the plant material. These specimens are typically readily identified by their characteristic de-colouration ('yellowing'), and were treated with caution in the dataset. Note that affected specimens would have lower apparent Ni, Co, Mn concentrations, and hence would not lead to 'false-positives' for detecting hyperaccumulator plants.

Even though the handheld XRF instrument can accurately measure Zn concentrations, and Zn has been measured previously in herbarium specimens [START_REF] Gei | A systematic assessment of the occurrence of trace element hyperaccumulation in the flora of New Caledonia[END_REF]Nkrumah et al., 2018a.b), Zn data has been excluded from this dataset because some specimens at the FRC Herbarium have been glued to the cardboard sheets with a glue that has high concentrations of Zn (>1 Wt%). The type of glue that is Zn-rich was used mainly on older specimens (pre-1980s), but the identity of specimens that were affected could not reliably be established.

Statistical analyses

The apparent limits of detection (LOD) for Ni, Co and were estimated by visual inspection of the logtransformed linear regression models of the XRF data against corresponding ICP-AES measurements and set at: <190 μg g -1 for Ni, <140 μg g -1 for Co and <336 μg g -1 for Mn. The residuals vs. fitted values were inspected for each linear regression analysis, and outliers (± 3 SD of the residual) were identified and removed. Secondary linear regression models were then derived after the samples with XRF values <LOD were removed. Herbarium specimens with XRF values < LOD were excluded from the dataset, and of the total 7337 specimens that were measured, 6775 specimens remained in the final dataset. The XRF-ICP-AES correction factors were then applied to all of the raw XRF measurements obtained from the herbarium specimens.

The data were analysed using Xlstat (Microsoft professional 2016) and comprised of computing range and means, and graphing plots. The phylogenetic data was analysed using the packages "ape" and "phylo4d" in R and the software PHYLOGENETIC (Phylomatic tree R20120829 Plants).

RESULTS

General statistics and phylogenetic patterns

Overall, 6775 specimens (representing 119 different families) originating from ultramafic and nonultramafic soils were analysed by XRF for Ni, Co and Mn and this yielded 759 specimens (in in 17 families, 30 genera and 74 species) as trace element hyperaccumulators. This includes 28 Ni hyperaccumulator species (in 10 families, 17 genera), 12 Co hyperaccumulator species (in 3 families, 7 genera), and 51 Mn hyperaccumulator species (in 12 families, 24 genera). Some specimens hyperaccumulated a combination of Ni, and/or Co and/or Mn simultaneously. All specimens hyperaccumulating Ni and Co originated from ultramafic areas, whereas Mn hyperaccumulators are widespread and very few specimens originated from known ultramafic areas. Hyperaccumulators of Ni, Co, and Mn in the Malpighiales (Table 1) and for other clades (Table 2), and hyperaccumulation records are broken down into families and genera. The Phyllanthaceae had the greatest number of hyperaccumulators among the families tested, with nine genera containing hyperaccumulators and four genera containing hyperaccumulators of all three elements. The second highest number of hyperaccumulating genera was the Salicaceae, with four genera, but only Ni and Mn were hyperaccumulated in this family. This study examined the accumulation patterns of Ni, Co and Mn in 24 families, 78 genera and 351 species from the Malpighiales, and a phylogenetic tree was constructed based on these results (Figure 1).

Nickel hyperaccumulation

In total, 271 specimens had Ni concentrations greater than the LOD (<190 µg g -1 ), of those 163 specimens exceeded hyperaccumulation threshold (>1000 µg g -1 ), with the highest value recorded in Dichapetalum gelonioides with 45 600 µg g -1 Ni. As such, only 4% of the specimens, had detectable Ni. The incidence of Ni hyperaccumulation is shown in Figure 3. A total of 10 families with 17 genera and 28 species were identified as Ni hyperaccumulators. Nickel hyperaccumulation was recorded in unrelated families, such as the Achariaceae (Hydnocarpus -2 spp.), Annonaceae (Marsypopetalum -1 sp.), Dipterocarpaceae (Shorea -1 sp.), Fabaceae (Dalbergia -1 sp.), Loganiaceae (Strychnos -1 sp.) and Meliaceae (Walsura -2 spp.), but is most frequent in the Phyllanthaceae (genera: Actephila, Antidesma, Aporusa, Ashtonia, Baccaurea, Cleistanthus Glochidion, Phyllanthus). Particularly interesting new records include an unidentified species of Hydnocarpus (Achariaceae) with up to 3200 µg g -1 Ni, Marsypopetalum pallidum (Annonaceae) with 6000 µg g -1 Ni. The rare dipterocarp Shorea tenuiramulosa first reported by [START_REF] Proctor | Ecological studies on Gunung Silam, a small ultrabasic mountain in Sabah, Malaysia. II. Some forest processes[END_REF] Antidesma montis-silam with 4600-32 700 µg g -1 Ni is a rare taxon principally known from Mt Silam on Sabah's east coast, which has been the subject of a recent field-based study (Nkrumah et al. 2017).

Other Ni hyperaccumulating species in this genus include A. neurocarpum (up to 21 500 µg g -1 Ni), A. riparium (up to 2900 µg g -1 Ni) and A. puncticulatum (up to 3500 µg g -1 Ni). The genus Aporosa also yielded several Ni hyperaccumulators, including A. lucida with up to 10 400 µg g -1 Ni (and up to 980 µg g -1 Co), all expect A. chalarocarpa were unknown previously. Numerous Ni hyperaccumulating species of Glochidion all of which were previously recorded, were also detected (see Suppl Info for details). Note that several Glochidion taxa from Mount Kinabalu Park (G. cf. mindorense, G. cf. sericeum, G. "panatanran", G. "bambangan", G. "Nalumad"). There are at the Sabah Parks Herbarium (SNP) and could not be matched with specimen sheets the FRC Herbarium, likely because there are yet undescribed taxa endemic to Mount Kinabalu. The genus Glochidion, which consists of more than 300 species mainly distributed in the Indo-Pacific region, is in desperate need of a modern revision for Borneo, with regional herbaria typically housing thousands of unidentified Glochidion specimens. The FRC Herbarium only has specimens of six Phyllanthus taxa: P. lamprophyllus, P. balgooyi (synonym P. hellwigii), P. kinabaluicus, P. rufuschaneyi (formerly P.

cf. securinegioides and identified as P. gracilipes), P. urinaria and P. amarus. The latter two a pantropical weeds, whereas P. lamprophyllus is a common species along rivers in Sabah. The other two taxa are hypernickelophores, whereas P. kinabaluicus is a hyper-endemic to Mount Kinabalu (and a Ni accumulator with 700 µg g -1 Ni, unpublished data).

In the large genus Psychotria (Rubiaceae) three taxa (P. densifolia, P. gracilis, P. sarmentosa) hyperaccumulate Ni, up to 24 500 µg g -1 , but it is uncertain whether these taxa are actually distinct The majority of Ni hyperaccumulators are 'obligate' (e.g. hyperaccumulate Ni consistently for all specimens), for example Phyllanthus rufuschaneyi with 25 samples ranging from 2196-23 300 µg g - 1 Ni (van der Ent et al. 2015f) or high but variable (but with some specimens below the Ni hyperaccumulation threshold i.e. >1000 µg g -1 Ni) such as Rinorea bengalensis with 45 specimens ranging from 250-32 200 (with a mean of 7400 µg g -1 Ni), or Walsura pinnata with 22 specimens 290-8000 ranging from 290-8000 (with a mean of 2300 µg g -1 Ni). Only a few Ni hyperaccumulators are probably truly 'facultative', such as Antidesma neurocarpum with 196 specimens ranging from 370-21 500 µg g -1 Ni (with a mean of 2900 µg g -1 Ni). However, for most other Ni hyperaccumulating species not enough specimens (most species were represented by less than 5 specimens) were available to draw any definitive conclusions about obligate or facultative hyperaccumulation status.

The frequency distribution of Ni concentrations in the herbarium specimens (Figure 2) is distinct from Co and Mn (see further below). There are two separate modes on ultramafic and non-ultramafic soils (confirmed by statistical tests shown in Table 3). On non-ultramafic soils the distribution of Ni follows a pseudo 'log-normal' distribution with a geometric mean of ~320 µg g -1 . On ultramafic soils, two groups exist, the first follows a log-normal distribution with a geometric mean of ~500 µg g -1 (e.g. below the Ni hyperaccumulation threshold), whereas the second group also follows a log-normal distribution and has a geometric mean of ~10 000 µg g -1 (e.g. 'hypernickelophores'). The two groups are clearly separated at ~1000 µg g -1 which is the recognised threshold to define Ni hyperaccumulation. Statistical tests reveal that Ni concentrations in specimens from ultramafic and non-ultramafic soils are significantly different (Table 3). Only the genus Baccaurea (Phyllanthaceae) and the family Salicaceae show no significant difference between non-ultramafic and ultramafic soils.

Cobalt hyperaccumulation

In total, 107 specimens had Co concentrations greater than the LOD (<140 µg g -1 ), of those 23 specimens exceeded the hyperaccumulation threshold (>300 µg g -1 ), with the highest value recorded in Ashtonia excelsa with 1500 µg g -1 Co. The frequency distribution shows that only a very limited number of specimens are clearly separated based on their concentrations (Figure 2). There were only three families, nine genera and nine species which had Co concentrations >300 µg g -1 (Table 1 and2). The Phyllanthaceae family emerged as the main Co (hyper)accumulator group. The known Co hyperaccumulator Glochidion cf. sericeum was not detected in this study, but that can be explained because the FRC Herbarium does not hold any specimens of this taxon, since they are deposited at the Sabah Parks Herbarium (van der Ent et al., 2018c). In the family Phyllanthaceae, samples originating from ultramafic soils tended to have higher foliar Co, notably in the genus Baccaurea (Table 3).

Manganese hyperaccumulation

In total 4218 specimens had Mn concentrations greater than the LOD (<336 µg g -1 ), of those 123 specimens exceeded the hyperaccumulation threshold (>10 000 µg g -1 ), with the highest value recorded in Antidesma puncticulatum with 46 480 µg g -1 Mn. As such, about one third of the specimens measured, originating from 46 families, had detectable Mn. Although hyperaccumulation of Mn is relatively rare, concentrations exceeding 550 µg g -1 are frequent in the flora of Sabah. A total of 12 families, 24 genera and 51 species were Mn hyperaccumulators (Tables 1 &2). The Phyllanthaceae had the greatest numbers of Mn hyperaccumulators with six genera and 27 species.

Manganese hyperaccumulation is clearly phylogenetically scattered, and in most instances just a single or a few specimens of a species were found with inordinately high Mn values. For example, Diospyros singapurensis (Ebenaceae) with 19 700 µg g -1 Mn, Chrozophora oblongifolia (Euphorbiaceae) with 11 600 µg g -1 Mn, Gymnacranthera sp. (Myristicaceae) with 10 200 µg g -1 Mn, and Syzygium fastigiatum (Myrtaceae) with up to 28 800 µg g -1 Mn. Such instances may be explained by local variations in soil Mn availability in which the particular specimen grew. In other cases, Mn hyperaccumulation is a more consistent trait with numerous specimens of the same species having extremely high values. Some of these are especially strong Mn hyperaccumulators, such as Casearia tuberculata with up to 38 200 µg g -1 Mn, Baccaurea sumatrana with up to 21 700 µg g -1 Mn, Neoscortechinia philippinensis (Euphorbiaceae) with three specimens with 3600-11 800 µg g -1 Mn, and Aporosa illustris with up to 26 600 µg g -1 Mn. The genus Antidesma is noteworthy for the numbers of Mn hyperaccumulating specimens, including A. neurocarpum with 192 specimens ranging from 370-13 900 µg g -1 Mn (mean of 2830 µg g -1 Mn), and A. tomentosum with 112 specimens ranging from 390-17 400 µg g -1 Mn (mean of 3190 µg g -1 Mn). The genus Walsura in the Meliaceae family is of similar interest because three taxa (W. decipiens, W. pachycaulon, W. pinnata) are strong Mn hyperaccumulators with up to 21 000 µg g -1 , Mn 45 200 µg g -1 Mn and 30 700 µg g -1 Mn respectively. In all species, however, the mean Mn value does not exceed the Mn hyperaccumulation threshold (i.e. >10 000 µg g -1 Mn). Nickel and Mn hyperaccumulation in Antidesma, focussing on A. montissilam, has been subject of a dedicated study (Nkrumah et al. 2017). Yet other taxa have very wideranging Mn values, for example Mischocarpus pentapetalus (Sapindaceae) with 51 specimens ranging from 650-19 400 µg g -1 Mn. Interesting, the related M. pinnata is a strong Ni hyperaccumulator. Another example is Cleistanthus sumatranus (Phyllanthaceae) with 54 specimens ranging from 350-11 300 µg g -1 Mn.

The frequency distribution of Mn concentrations in the herbarium specimens (Figure 2) shows that almost all the specimens fall in a log-normal distribution with a geometric mean of 1000 µg g -1 . There does not seem to be an effect of ultramafic soils in the pattern of foliar Mn concentrations. However, statistical differences between specimens from the Violaceae family from ultramafic and nonultramafic soils were found (p <0.05) (Table 3). The Violaceae originating from non-ultramafic soils had Mn concentrations higher than the Violaceae originating from ultramafic soils (p <0.01). Also, the Phyllanthaceae had more Mn hyperaccumulator species growing on non-ultramafic soils than on ultramafic soils.

Simultaneous hyperaccumulation of two or more elements

The genus Baccaurea, and especially B. odoratissima with up to 8000 µg g -1 Ni, is of interest because of its ability to also accumulate Co up to 960 µg g -1 . Ashtonia excelsa, with only one taxon in Borneo, also stands out because it accumulates up to 8100 µg g -1 Ni and 1500 µg g -1 Co. Walsura pinnata (Meliaceae) is another taxon that can hyperaccumulate Ni (up to 8000 µg g -1) , Co (up to 4580 µg g -1 in van der Ent et al. 2015b) and Mn (up to 30 700 µg g -1 ). Rinorea bengalensis and R. javanica incidentally also have high Co values, with up to 630 µg g -1 and 670 µg g -1 respectively [START_REF] Brooks | Detection of nickeliferous rocks by analysis of herbarium specimens of indicator plants[END_REF].

Hyperaccumulation of Co is relatively rare, mostly because soils with high available Co are infrequent [START_REF] Lange | Copper and cobalt accumulation in plants: a critical assessment of the current status of knowledge[END_REF]van der Ent et al., 2018c, d). In this study, only a few specimens from the same genera in the Phyllanthaceae hyperaccumulated Co alone. Other species identified in this study as Co hyperaccumulators also hyperaccumulated Ni and Mn simultaneously. Furthermore, in most ultramafic soils worldwide, Co correlates with Ni (van der [START_REF] Van Der Ent | Multi-element concentrations in plant parts and fluids of Malaysian nickel hyperaccumulator plants and some economic and ecological considerations[END_REF]Tashakor et al., 2018;van der Ent et al., 2018c,d).

New hyperaccumulator plant discoveries

The herbarium XRF scanning in this study led to the recording of a total of 28 Ni hyperaccumulator species from 10 families and 17 genera (Suppl. Table 2). Among the Ni hyperaccumulators were 10 species that can be classified as 'hypernickelophores' of which eight were already known prior to this study [START_REF] Baker | Hyper accumulation of nickel by the flora of the ultramafics of Palawan, Republic of the Philippines, The vegetation of ultramafic serpentine[END_REF]van der Ent et al., 2015bvan der Ent et al., , 2016c;;[START_REF] Bouman | Phyllanthus rufuschaneyi: a new nickel hyperaccumulator from Sabah with potential for tropical phytomining[END_REF]Nkrumah et al., 2018a). The 'hypernickelophores' were from the Dichapetalaceae, Phyllanthaceae, Violaceae and Rubiaceae, and the two newly reported hypernickelophores were from the genus Xylosma (Salicaceae); Xylosma luzoniensis and X. sumatranum. Moreover, the recently reported hypernickelophore Antidesma montis-silam was discovered through herbarium XRF scanning and plant material was subsequently collected in the field and analysed, confirming the findings from the XRF survey (Nkrumah et al., 2018a). The present study found that the highest Ni concentrations in herbarium specimens in Dichapetalum gelonioides subsp. tuberculatum with 4.56 Wt% Ni. This taxon was already the species with the highest Ni concentration previously reported with 2.7 Wt% (Nkrumah et al. 2018b). Dichapetalum gelonioides subsp. sumatranum and D. gelonioides subsp.

pilosum are strong Zn hyperaccumulators on normal soils, reaching up to 15 700 µg g -1 and 26 400 µg g -1 foliar Zn [START_REF] Baker | Hyper accumulation of nickel by the flora of the ultramafics of Palawan, Republic of the Philippines, The vegetation of ultramafic serpentine[END_REF]Nkrumah et al., 2018b), and although extremely high Zn were detected in this study in Dichapetalum spp. (and Zn-rich glue could be eliminated in these cases after careful inspection of the specimens), we omitted Zn values from the overall dataset for consistency (as the presence or absence of the problematic glue could not be established in all cases). This study also recorded a total of 51 Mn hyperaccumulator species from 13 families and 24 genera (Suppl. Table 3), of which 33 were previously known to be hyperaccumulators (van der Ent et al., 2018d). The present record for Mn hyperaccumulation in Sabah is Antidesma puncticulatum with 4.6 Wt% Mn (Nkrumah et al. 2017), followed by Walsura pachycaulon with 4.5 Wt% Mn, and Casearia tuberculata with 3.8 Wt% Mn. Additionally, 14 Co hyperaccumulator species from three families and eight genera (Suppl. Table 4) were found, of which seven were previously known to be hyperaccumulators (van der Ent et al., 2018d). The highest Co concentration in herbarium specimens was recorded in Ashtonia excelsa with 1500 µg g -1 foliar Co.

DISCUSSION

Hyperaccumulator plants have been studied from many different angles for their unusual biochemistry and ecophysiology [START_REF] Baker | Terrestrial Higher Plants Which Hyperaccumulate Metallic Elements[END_REF]van der Ent et al., 2017), as well as for their potential utility in agromining applications (van der Ent et al., 2013b(van der Ent et al., , 2015a)). The identification of new hyperaccumulators can provide information about the evolution of hyperaccumulation in the Plant Kingdom. At the global scale Ni hyperaccumulator plants are known primarily to occur in the Brassicales (in the northern hemisphere temperate and Mediterranean climates) and Malpighiales (in tropical climates of the Old and New World) (Reeves, 1992[START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF][START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF]. Although Ni hyperaccumulation has been most intensively studied at global scale [START_REF] Reeves | A global database for hyperaccumulator plants of metal and metalloid trace elements[END_REF], as well as in the Sabah (van der Ent et al., 2018), this study led to the discovery of numerous hyperaccumulators for Mn and several for Co. This places Sabah among the top regions in the world for the diversity of Ni hyperaccumulators after Cuba with 120 species [START_REF] Reeves | The flora and biogeochemistry of the ultramafic soils of Goiás state, Brazil[END_REF] and New Caledonia with 65 species [START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF]. This study also confirmed that Ni hyperaccumulation is edaphically restricted to ultramafic soils and a phylogenetic proponency for the Malpighiales. In these respects, Ni hyperaccumulation is therefore a distinct phenomenon from Mn hyperaccumulation.

This study led to the recording of 51 hyperaccumulator species and hence added significantly to the global inventory of Mn hyperaccumulator plants (only 14 Mn hyperaccumulator species were previously known from Sabah). These Mn hyperaccumulator species are phylogenetically scattered and occur, amongst others, in the Celastraceae, Clusiaceae, Euphorbiaceae, Meliaceae, Phyllanthaceae, Salicaceae and Violaceae. Manganese is not specifically accumulated in plants occurring on ultramafic soils, in stark contrast to Ni (and Co), although ultramafic soils in Sabah are significantly higher in Mn than other 'normal' soils (van der Ent et al., 2017a). Given that Mn hyperaccumulation is phylogenetically widely distributed, and not associated with ultramafic soils, it is likely it is far more common than previously thought [START_REF] Reeves | Hyperaccumulation of trace elements by plants[END_REF]. There are at least two mechanisms response for Mn hyperaccumulation: (i) rhizosphere acidification from the release of carboxylic acids (i.e. citrate) by roots for P uptake which can induce Mn accumulation [START_REF] Lambers | Leaf manganese accumulation and phosphorusacquisition efficiency[END_REF], as shown for Lupinus albus and Phytolacca americana [START_REF] Jiang | Biomass and metal yield of co-cropped Alyssum murale and Lupinus albus[END_REF][START_REF] Degroote | Interactions of the manganese hyperaccumulator Phytolacca americana L. with soil pH and phosphate[END_REF], and (ii) resulting from the mineralogical changes of Mn oxides in soils during oxidation/reduction cycles. Soil reduction results in dissolution of Mn oxides and increased availability of Mn under the Mn 2+ ion. This mechanism has been identified as a cause for Co hyperaccumulation in Stagnosols by facultative Co hyperaccumulators (Co is extremely 'manganesophilic' in soils) [START_REF] Lange | Copper and cobalt accumulation in plants: a critical assessment of the current status of knowledge[END_REF].

This study also led to the recording of 12 Co hyperaccumulator species from three families and seven genera. Seven Co hyperaccumulator species were already known previously [START_REF] Reeves | Hyperaccumulation of trace elements by plants[END_REF]van der Ent et al., 2015bvan der Ent et al., , 2018c, d), d), and those recorded in this study occur in the Meliaceae (genus Walsura), Phyllanthaceae, and Violaceae (genus Rinorea). The highest Co concentration was found in Ashtonia excelsa with 1500 µg g -1 . The results of this study confirmed that Co hyperaccumulation is relatively rare, and that some taxa in the Phyllanthaceae (e.g. Baccaurea membranacea) can accumulate both Co and Ni at concomitantly high concentrations.

Finally, the outcomes of this research demonstrate that handheld XRF is highly useful approach for hyperaccumulator plant discovery in herbarium collections that has the potential to add vast numbers of hyperaccumulating taxa to the global inventory. This research represents the largest single contribution to the global knowledgebase on hyperaccumulator plants, and includes a substantial expansion in phylogenetic records, many of the recorded genera being previously unknown to contain hyperaccumulator plants. This reiterates the importance of systematic screening across different families, as undertaken in this study, to avoid a bias. The current main limitation is the lack of sensitivity for transition elements present at concentrations < 300 µg g -1 and for light elements (elements with a Z <20 such as Al). However, technological advances, including monochromatically excited XRF, will enable higher sensitivity for most elements of the Periodic Table . Newly discovered hyperaccumulators may eventually be used for genetic and ecophysiological experiments under controlled conditions to advance our fundamental understanding of trace element hyperaccumulation [START_REF] Gei | Tools for the Discovery of Hyperaccumulator Plant Species and Understanding Their Ecophysiology in[END_REF]Nkrumah et al., 2018a). The discovery of hypernickelophores (>1 Wt% Ni) is promising for future advances in the development of Ni agromining. The two species of Xylosma discovered in this study are fast growing trees and might prove as useful as other species that are currently being used in agromining in Sabah, such as Phyllanthus rufuschaneyi [START_REF] Bouman | Phyllanthus rufuschaneyi: a new nickel hyperaccumulator from Sabah with potential for tropical phytomining[END_REF]. 1. Co, Mn and Ni hyperaccumulator genera from the Malpighiales. The number of specimens, ranges and means above the hyperaccumulator threshold are shown. "-" is below the element-specific hyperaccumulation threshold (>300 µg g -1 Co, >1000 µg g -1 Ni, >10 000 µg g -1 Mn). Notes: 1 number of specimens from unidentified species from this study between brackets.

2 number of hyperaccumulator specimens from this study between parentheses. 3 number of hyperaccumulator specimens from unidentified species from this study between brackets.

Table 2. Co, Mn and Ni hyperaccumulator genera from Orders (excluding Malpighiales). With the number of specimens and foliar ranges above the hyperaccumulator threshold. "-" is below the element-specific hyperaccumulation threshold (>300 µg g -1 Co, >1000 µg g -1 Ni, >10 000 µg g -1 Mn). Notes: 1 number of hyperaccumulator specimens from this study between parentheses. 2 number of hyperaccumulator specimens from unidentified species from this study between brackets.

Table 3. Families and selected genera found on ultramafic and non-ultramafic substrates showing the corrected XRF values of their ranges, mean values, the total number of specimens and t-test (pvalue at 0.05). "-" is below the element-specific LOD (<190 μg g -1 Ni, <140 μg g -1 , <336 μg g -1 Mn). *values from all the genera in the family. Concentration scales (X-axis) log10 transformed and actual values in µg g -1 . Numbers of specimens on Y-axis. Dotted line denotes the element-specific hyperaccumulation threshold (>300 µg g -1 Co, >1000 µg g -1 Ni, >10 000 µg g -1 Mn).

  from Mt Silam on Sabah's east coast as a Ni hyperaccumulator was not detected in this survey, but it is a weak Ni hyperaccumulator with up to 1790 µg g -1 Ni (van der Ent and Mulligan 2015). Values for Dichapetalum gelonioides (Dichapetalaceae) are extremely wide-ranging (340-45 600 µg g -1 Ni), which may be explained by the omission of infraspecific ranks in this study. The various Dichapetalum subspecies differ greatly in their hyperaccumulation characteristics, for example subsp. sumatranum and pilosum do not accumulate Ni, but Zn (Nkrumah et al. 2018b). Some known Ni hyperaccumulator plants species, such as Kibara coriacea (Monimiaceae) with up to 5840 µg g -1 Ni (van der Ent and Mulligan 2015) and Actephila alanbakeri (Phyllanthaceae) with up to 14 700 µg g -1 Ni (van der Ent et al. 2016) were not detected because the FRC Herbarium does not hold any specimens of these taxa. This survey added another species of Actephila, A. excelsa, as a strong Ni hyperaccumulator with 5200-14 600 µg g -1 Ni. Another such instance is the known Ni hyperaccumulator Walsura pinnata (Meliaceae) which can accumulate up to 8000 µg g -1 Ni, with the new recording W. pachycaulon with 690-8600 µg g -1 Ni.

  given the taxonomical uncertainties in this genus. The large family Sapindaceae, with the well-known fruits Nephelium lappaceum (Rambutan) and Dimocarpus longan (Longan), only yielded one Ni hyperaccumulator in Mischocarpus sundaicus, which was previously known and can accumulate up to 4420 µg g -1 Ni (van der Ent et al. 2015f), although lower values up to 2400 µg g -1 Ni were recorded with XRF analysis. The Violaceae (genus: Rinorea) has two Ni hyperaccumulating taxa (R. bengalensis, R. javanica) which were previously known and again detected in this study. They can accumulate up to 22 200 µg g -1 and 9680 µg g -1 Ni respectively (van der Ent et al. 2017a). The Salicaceae has four genera (Flacourtia, Homalium, Scolopia, Xylosma) with Ni hyperaccumulators. Of these two unidentified Scolopia spp. (likely S. luzonensis) accumulates up to 9200 µg g -1 Ni, whereas S. spinosa accumulates 1240 µg g -1 Ni.

FIGURE CAPTIONS Figure 1 .

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Figure 2 .

 2 Figure 2. Frequency distribution of Ni, Co and Mn elemental concentrations in herbarium specimens originating from ultramafic (shaded blue) and non-ultramafic soils (shaded grey).

Figure 3 .

 3 Figure 3. Phylogenetic tree of Ni, Co and Mn hyperaccumulator taxa in the flora of Sabah. The tree was constructed using PHYLOGENETIC (Phylomatic tree R20120829 Plants) and shows orders, clades. families and genera with hyperaccumulators.

TABLE CAPTIONS Table

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ACKNOWLEDGMENTS

We thank Sabah Parks for their support and the Sabah Biodiversity Council (SaBC) for granting permission for conducting research in Sabah. The University of Queensland is acknowledged for financial support that made this project possible. The French National Research Agency through the national "Investissements d'avenir" program (ANR-10-LABX-21, LABEX RESSOURCES21) and through the ANR-14-CE04-0005 Project "Agromine" is acknowledged for funding support. A. van der Ent was the recipient of a Discovery Early Career Researcher Award (DE160100429) from the Australian Research Council. A. Ocenar was the recipient of an Australia Awards PhD Scholarship from the Australian Federal Government.