Organocatalytic acetylation of pea starch: Effect of alkanoyl and tartaryl groups on starch acetate performance

Organocatalytic acetylation of pea starch was systematically optimized using tartaric acid as catalyst. The effect of the degree of substitution with alkanoyl (DSacyl) and tartaryl groups (DStar) on thermal and moisture resistivity, and film-forming properties was investigated. Pea starch with DSacyl from 0.03 to 2.8 was successfully developed at more efficient reaction rates than acetylated maize starch.

Available online 27 June 2022

0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Organocatalytic acetylation of pea starch: Effect of alkanoyl and tartaryl

groups on starch acetate performance

aCenter for Innovative Food (CiFOOD), Department of Food Science, Aarhus University, AgroFood Park 48, Aarhus N 8200, Denmark

bAarhus Institute of Advanced Studies (AIAS), Aarhus University, DK-8000 Aarhus, Denmark

cSchool of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China

A R T I C L E I N F O

Keywords:

Organocatalytic esterification

Tartaric acid

NMR

Packaging

Biofilms

Chromatography

A B S T R A C T Organocatalytic acetylation of pea starch was systematically optimized using tartaric acid as catalyst The effect

of the degree of substitution with alkanoyl (DSacyl) and tartaryl groups (DStar) on thermal and moisture re-sistivity, and film-forming properties was investigated Pea starch with DSacyl from 0.03 to 2.8 was successfully developed at more efficient reaction rates than acetylated maize starch Nevertheless, longer reaction time resulted in granule surface roughness, loss of birefringence, hydrolytic degradation, and a DStar up to 0.5 Solid- state 13C NMR and SEC-MALS-RI suggested that tartaryl groups formed crosslinked di-starch tartrate Acetylation increased the hydrophobicity, degradation temperature (by ~17 %), and glass transition temperature (by up to

~38 %) of pea starch The use of organocatalytically-acetylated pea starch with DSacyl ≤0.39 generated starch- based biofilms with higher tensile and water barrier properties Nevertheless, at higher DS, the incompatibility between highly acetylated and native pea starches resulted in a heterogenous/microporous structure that worsened film properties

1 Introduction

Pea starch is, more than ever before, an abundant by-product from

the increasing production of protein ingredients from field peas,

repre-senting an inexpensive, non-toxic, and annually renewable starch source

Unfortunately, pea starch demand does not match its escalating

abun-dance due to its inherent properties As any other starch, pea starch has

several limitations as a replacer of fossil-based polymeric materials,

slow recrystallisation after processing that leads to the progressive

resulting in poor moisture sensitivity Furthermore, the inherent water

content of starch can lead to considerable hydrolysis and molar mass

decrease during processing (Imre & Vilaplana, 2020) This deserves

special consideration in those starches with elevated relative proportion

of B-type crystalline polymorphism, such as pea, which possesses 22–55

% of crystals found as B-type allomorph and, hence, with a central cavity

Bertoft, 2010; Ren et al., 2021) Pea starch also has the typical

limita-tions of most starches as a food ingredient or drug excipient, such as poor

stability and processing tolerance, high water sorption, low shear, and

Shog-ren, 1996; Singh et al., 2007), and additional limitations due to its amylose-driven shortcomings, including low and slow granular swelling

Interestingly, the three hydroxyl groups in C2, C3 and C6 in the anhydroglucose units from starch, which confer the hydrophilic nature

to the molecule, are available to be chemically esterified with carboxylic acids, or carboxylic acid anhydrides or chlorides Starches having a low degree of substitution (DS, average number of hydroxyls replaced by other moieties per repeating unit) find numerous applications in the food industry as adhesive, thickening, texturizing, stabilizing, and

Vilaplana, 2020; Ragavan et al., 2022) Moreover, starch esterified with short chain fatty acids (e.g., acetate, propionate, butyrate) have the

2003; Clarke et al., 2011; Nielsen et al., 2019) According to the EU

Regulation (EC) No 1333/2008 (2008), acetylated starch is listed as food additive (E1420) and can present a maximum level of acetyl groups of

* Corresponding author

E-mail address: mm@food.au.dk (M.M Martinez)

Carbohydrate Polymers

https://doi.org/10.1016/j.carbpol.2022.119780

Received 23 February 2022; Received in revised form 20 May 2022; Accepted 22 June 2022

corresponds to a DS of 0.097 On the other hand, esterified starches with

intermediate (0.2–1.5) and high DS (1.5–3.0) can be used as

thermo-plastic materials with improved thermal stability and reduced moisture

Currently, commercial starch esters are produced using carboxylic

acid anhydrides and sodium hydroxide (NaOH) as catalyst in aqueous

Elomaa et al., 2004; Singh et al., 2004; Xu et al., 2004) However, this

starch esterification raises some environmental and safety concerns

since a large volume of wastewater and sodium acetate are generated

(Aˇckar et al., 2015; Ragavan et al., 2022) In this sense, the use of green

organocatalysts with controlled catalytic performance has emerged as a

possible efficient solution to replace NaOH A wide variety of

organo-catalysts, such as amino acids and hydroxy acids (e.g., lactic, citric, or

tartaric acids), are naturally available from biological sources as single

enantiomers with controlled catalytic performance This leads to several

remarkable applications in solvent-free and metal-free conditions ideal

to modify biopolymers for food packaging, food, pharmaceutical and

can be produced at large scale by biotechnological routes in a

therefore, are cheap to prepare and readily accessible in a range of

quantities suitable for industrial-scale reactions Last but not least,

natural organocatalysts are insensitive to oxygen and moisture in the

atmosphere, so there is no need for special reaction equipment and

experimental techniques, and are fully biodegradable, non-toxic and

Imre and Vilaplana evidenced that, among many organocatalysts,

the hydroxycarboxylic tartaric acid, followed by citric acid, exhibited a

relevant catalytic effect in maize starch esterification (Imre & Vilaplana,

cata-lyze the esterification of starch with several 1-substituted

mono-carboxylic acid and anhydride derivatives of n-alkanes, including acetic,

2016; Tupa et al., 2013, 2015; Nielsen et al., 2018) It must be noted that

in these studies the substrate used was maize starch Remarkably, the

apparent recalcitrance of native maize starch was greatly influenced by

the role of amylose in stabilizing the semi-crystalline structure of maize

starch and restricting granular swelling (Imre & Vilaplana, 2020; Luo &

Shi, 2012) Although pea starch presents lower granular swelling, less

porous granular structure, higher proportion of B-type crystalline

polymorphism, and considerably smaller amylopectin compared to

orga-nocatalytic derivatization has never been studied

For the first time, we systematically report the organocatalytic

acylation of pea starch using tartaric acid as catalyst We hypothesize

that pea starch exhibits lower recalcitrance towards organocatalytic

esterification than maize starch, and that both alkanoyl and tartaryl

of the resulting starch acetates Pea starch acetates were studied in terms

of chemical structure, molecular weight, granular morphology,

crys-tallinity, and thermal properties Moreover, we investigated the role of

the developed tartaric acid-catalyzed pea starches on the mechanical,

thermal and water barrier properties of pea starch-based biofilms

2 Materials and methods

2.1 Materials and reagents

Commercial starch from smooth pea was gently provided by Cosucra

Group Warcoing S.A (Warcoing, Belgium) Maize starch was purchased

from Ingredion Inc (Bridgewater, NJ, USA) Pea and maize starch were

freeze-dried for 24 h prior to use to avoid the interference of moisture in

the acetylation process

Analytical grade acetic anhydride, L-(+)-tartaric acid (>99 %

purity), and lithium bromide were purchased from Sigma Aldrich (Søborg, Denmark) Hydrochloric acid, analytical grade ethanol, sodium hydroxide, phenolphthalein, and glycerol were obtained from VWR in-ternational (Søborg, Denmark)

2.2 Organocatalytic acetylation

The organocatalytic acetylation of pea starch was performed following other studies focusing on maize starch, such as those from Imre & Vilaplana and Tupa et al with some modifications (Imre & Vilaplana, 2020; Tupa et al., 2015) The pea starch: tartaric acid ratio

acetic anhydride (0.52 mol) in a 100 mL round flask with a magnetic stirrer and a reflux condenser to avoid the loss of acetic anhydride An

oil bath When completely dissolved (after 15 min), the temperature was increased to the desired reaction temperature (85, 95, 110, and

ranging from 30 min to 8 h were tested to evaluate the effect of this parameter on the degree of substitution of pea starch After the reaction, the dispersed mixture was cooled down at room temperature and the solid material separated from the solvent by vacuum filtration in a Buchner funnel with Whatman No 1 filter paper To ensure the complete removal of the solvent and organocatalyst, 3 washes with distilled water and 1 with 50 % ethanol were performed Washed acetylated starch was

into powder to remove potential aggregates of starch granules Maize starch was used as a control to compare with pea starch for the kinetics

of the esterification reaction Samples were kept under controlled rela-tive humidity in a desiccator at room temperature until further analysis

2.3 Determination of the degree of esterification by chemical titration

Back titration with HCl was used to determine the acyl content and degree of substitution of starch acetates following the procedure from

Tupa et al (2015) Briefly, 0.11 g of acetylated starch was dispersed in

sus-pensions was decreased with 0.1 N NaOH and phenolphthalein was used

as indicator Subsequently, 20 mL of 0.1 N NaOH was added prior

were left under continuous stirring at room temperature for 48 h After this time, solutions were back titrated with 0.1 M HCl using the same indicator Native pea starch was used as the control The acyl content and degree of substitution (DS) were determined as follows:

Acyl (%) =(Vc − Vs)*0.1*M acyl*10− 3

weight of the acetyl groups (43.05 g/ mol), W is the weight of the dried

(162.14 g/ mol) Reported DS values were the mean of at least 2 repetitions

2.4 Determination of the chemical structure of starch acetates 2.4.1 Fourier Transform Infrared Spectroscopy (FTIR)

Dried starch acetates were analyzed in a Nicolet iS5 Fourier Trans-form Infrared Spectrophotometer (ThermoScientific, Denmark) attached to an iD5 Attenuated Total Reflectance (ATR) accessory (ThermoScienfitic, Denmark) ATR-FTIR was interfaced to a personal

computer operating under OMNIC 9 software (version 2.11) FTIR

spectra of native and esterified starch were acquired between 400 and

4000 cm− 1 at a resolution of 4 cm− 1 using 32 co-added scans The

assignment of the bands to the specific functional group vibration mode

2.4.2 Nuclear magnetic resonance

2.4.2.1 Proton Nuclear Magnetic Resonance ( 1 H NMR) Native and

acetylated starch (10 mg) were dissolved in deuterated dimethyl

to 5 mm NMR tubes Samples were analyzed on a Bruker Avance III NMR

operating at 600.13 MHz The experimental conditions were spectral

width, 5000 Hz; relaxation delay, 5 s; number of scans, 64; pulse width,

prepared before each analysis Residual non-deuterated DMSO signal at

2.549 ppm was used as a reference The spectra obtained were analyzed

using MestReNova software (version 14.2.1) (Mestrelab Research S.L.,

Santiago de Compostela, Spain) The acetylation degree of pea starch

acetates was calculated by using the ratio of the signal area of the

and the area of the signals between 3.2 and 5.5 ppm representing the

DS = A acyl

/

7

A Glc /3=

3*A acyl

Aacyl and AGlc were normalized according to the number of protons (3

and 7, respectively) contributing to the area of the spectral signals

2.4.2.2 Solid state SP and CP/MAS 13 C magnetic resonance Native and

spectro-scopic experiments were performed in a Bruker Avance 400 NMR

respectively Solid State Single Pulse (SP) and Cross-Polarization Magic

Angle Spinning (CP/MAS) NMR experiments were recorded at 300 K A

CP/MAS probe for 4 mm rotos using a spin-rate of 9 kHz, a radio

decoupling was applied Recycle delays of 16 and 128 s were used for CP

and SP/MAS, respectively Determination of the degree of substitution

was done by calculating the ratio of the areas of the signal due to the

carbon of the acyl group bonded to the carbonyl group (16.56 ppm) or to

the carbonyl carbon in the acid/ester (166.88 ppm), respectively, to that

of the glucose anomeric carbon of the anhydroglucose units in the starch

MAS NMR due to the different longitudinal relaxation times of the

2018)

2.5 Microstructure and birefringence

A small amount of pea starch samples was placed on a glass

micro-scope slide with a drop of water and covered by a cover slip Crystalline

and granular structure, as well as birefringence, of the native and

modified starches were observed by an optical microscope (Leica

Microsystems, Wetzlar, Germany) linked to an Infinity 3 camera and

controlled by the Infinity Analyze 6 software (version 6.0.0., Teledyne

Lumenera, Ontario, Canada) Images were captured with and without

polarized light with a 20× objective

2.6 X-ray powder diffraction patterns

The powder X-ray diffraction pattern of the samples were analyzed using a Bruker D8 Discover A25 diffractometer (Bruker AXS, Rheinfel-den, Germany) equipped with a copper tube operating at 40 kV and 30

mA, producing CuKa radiation of 0.154-nm wavelength The

of 0.02◦

2.7 Molecular size distribution and weight average molecular weight (M w )

Molecular weight distribution of fully branched native and acety-lated starch was determined following the procedures described by (Martinez et al., 2018) and (Roman et al., 2019) Briefly, 8.0 ± 0.5 mg of

a thermomixer (Eppendorf, Hamburg, Germany) at 350 rpm for 24 h Samples were then centrifuged at 4800 rpm for 15 min and the super-natant was collected Starch was precipitated with 10 mL 95 % ethanol,

and resuspended in 1.5 mL of DMSO containing 0.5 % lithium bromide

centrifugation (7000 rpm, 10 min), the supernatant was transferred to a vial for further analysis by High Performance Size Exclusion Chroma-tography (HPSEC, Agilent 1260 Infinity II, Agilent Technologies, Waldbronn, Germany) connected to a Multi-Angle Laser-Light Scat-tering detector (MALS) (Wyatt Technology, Santa Barbara, CA) and a refractive-index (RI) detector (Shodex RI-501, Munich, Germany) An

performed in GRAM 3000 and GRAM 30 (PSS GmbH, Mainz, Germany) columns connected in series Data to calculate weight average molecular

Technology, Santa Barbara, CA) using a second-order Berry plot pro-cedure The specific refractive index increment (dn/dc) was set as 0.066

et al., 2019) and second viral coefficient (A2) was assumed to be negligible

2.8 Thermal properties

Thermal properties of native and acetylated starch samples in dry

state (moisture content <2 % in all the samples) were investigated by

differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) DSC thermograms were obtained in nitrogen using a DSC Q2000 (Thermal Advantage Instruments-Waters LLC, USA) Dried starch (6 mg) was placed in an aluminum pan and hermetically sealed All the samples were subjected to heating/cooling cycles in a temperature range of

second heating run to eliminate any thermal history in the samples Thermal stability in the native and acetylated starch was evaluated using

a thermogravimetrical analysis system TGA-2 STARe from Mettler Toledo (USA) equipped with STARe software 5–6 mg of sample were placed in Mettler Toledo aluminum crucibles with punctured lids and

nitrogen environment The first derivative analysis (DTG) was per-formed and the peak temperature at which the thermal decomposition occurred was determined

2.9 Biofilm-making and properties

Starch-based biofilms were developed by the solvent casting method

native starch (1:3 acetylated:native starch ratio, w/w) due to the poor

solubility of highly acetylated starch Briefly, 4.0 g of starch (or the

native:acetylated starch blend) was mixed with 100 mL of deionized

water containing 25 % of glycerol (w/w of total solids) as the plasticizer

The film-forming solution was prepared by incubating the mixture at

gela-tinization of the starch and the good homogenization of the solution, the

(JULABO, SW22, Germany)

After incubation, the solution was cooled down at room temperature

for 30 min under gentle stirring and degassed using an ultrasonic bath

for 10 min Then, 40 mL of film-forming solution was gently poured into

for 15 h under circulating air After this time, biofilms were manually

peeled off from the petri dishes and kept in a desiccator with saturated

until further analysis

2.9.1 Biofilm thickness and mechanical properties

A portable digital dial pipe gauge (Diesella, Denmark) with the

ac-curacy 0.01 mm was used to measure the thickness of the films Eight

measurements were randomly taken at different points on each film

specimen The mechanical properties of the native and acetylated

starch-based biofilms were tested at room temperature in a Texture

Analyzer (Mecmesin, FTC, USA) with a 500 N load cell following ASTM

rectangular strips of 80 × 20 mm The strip was clamped between film-

extension grips (MECMESIN 500 N wedge grips, KYOCERA UNIMERCO

Tooling A/S, Denmark) which were set 50 mm apart The stretching

speed was 10 mm/min Force-distance curves were obtained and

transformed into stress-strain curves which allowed tensile strength at

break (TS, MPa), percentage of elongation at break (EB, %) and elastic

modulus (EM, MPa) to be obtained Mechanical properties were

calcu-lated as the average of eight repetitions

2.9.2 Thermal stability and gas barrier properties

Thermal stability of the biofilms was evaluated using a

thermogra-vimetrical analysis system TGA-2 STARe from Mettler Toledo (USA)

starches The water vapor permeability (WVP) of the film was

deter-mined by a permeability analyzer linked to a pressure-modulated

F1249-13 (2013) (23 ◦C and 85 % relative humidity, RH) following

initially preconditioned with the carrier gas (i.e., nitrogen 97.9 %

pu-rity) Then, the test gas (i.e., water vapor) was flushed To remove any

influence caused by film thickness differences, transmission rate values

was performed at least in duplicates for each film

2.10 Statistical analysis

Statistical analysis was conducted using XLSTAT premium software (version 2021.1.1, New York, USA) Differences among the starch and biofilm samples parameters were studied by analysis of variance

(ANOVA) When significant differences (p < 0.05) were found, Fisher's

Least Significant Difference (LSD) posthoc test was used to assess the

Re-lationships between degree of substitution in acetylated pea starch and starch or biofilm properties were performed by Pearson's correlation

3 Results and discussion

3.1 Optimization of the tartaric acid catalyzed acetylation process

With a view to synthesizing tartaric acid-catalyzed acetylated pea starch with different degree of substitution (DS), reaction temperature and time were systematically tested for DS values measured by the classical titration method The type of reagent (i.e., acetic acid anhy-dride) and catalyst (i.e., tartaric acid) were selected based on their high reactivity and catalytic efficiency, respectively, as shown in previous

Imre & Vilaplana, 2020; Tupa et al., 2013) However, temperature showed a higher acylation effect than reaction time More specifically, a

0.05 and 0.09, respectively An increase in the temperature to 95 and

were further increased with a longer reaction time (8 h) to 0.41 and

starches with low (≤0.2) and even intermediate DS (0.2–1.5) can be

substituted (DS < 0.2) granules only occurs in the amorphous parts of

the amylopectin fraction exclusively present in the outer lamella of the granules, as reported elsewhere for base-catalyzed smooth pea

Voragen, 2007b)

To achieve higher substitution levels, the reaction temperature was

ranging from 30 min to 4 h The reactivity significantly raised, reaching 0.39 DS in the first 30 min Furthermore, DS dramatically increased up

to 2.83 after 2 h of reaction, with a minimal improvement achieved after

3 h These findings align with those from Tupa et al., who attained DS of

et al., 2021) These results agree with other studies showing significantly

et al., 2013) Likewise, Imre & Vilaplana reported maximum DS of 2

Table 1

Weight average molecular weight (Mw) and degree of substitution of acyl (DSacyl) and tartaryl groups (DStar) at 135 ◦C calculated by back titration, 1H NMR, and Single- Pulse 13C NMR (SP 13C NMR)

Time (h) DS total DS acyl DS acyl DS tar DS total Starch acetate (%) Starch tartrate (%) Mw (10 5 g/mol)

*DSacyl and DStar were determined as described in Section 2.4.2 Starch tartrate DS (DStar) was calculated by the difference between DStotal and DSacyl values

even at 130 ◦C for 8 h (Imre & Vilaplana, 2020) The higher reactivity of

pea starch compared to maize starch could be explained by the different

effect of temperature on granule microstructure over the course of starch

derivatization, and, consequently, on granular reaction locale Smooth

pea starch exhibits lower gelatinization temperature attributed to the B-

initiated from the central hilum of starch granules, and the B-type

allomorph possesses a lower melting temperature than A-type

Furthermore, the absence of amylose lipid complexes of pea starch, as

opposed to normal maize starch, results in a significantly lower onset

this regard, some granular swelling has been deemed necessary to

a function of the properties of the reaction medium, but of the

temperature-driven dissociation of double-helical crystallites, loss of

crystallinity and, eventually, promotion of granule swelling, which

presumably is more likely to occur at shorter times in C-type granules,

such as those from smooth pea, at those high temperatures to attain high

DS

3.2 Degree and nature of substitution by spectroscopic techniques

The degree of substitution analyzed by chemical titration does not

distinguish between carboxylic acid species and related side groups, e.g.,

tartaryl side groups, which also results in overestimation of the DS of

acetylation reaction, not only the anhydride reagent can participate in

the substitution reaction, but also the dicarboxylic acid catalyst can

react to form starch tartrates, as illustrated in Fig 2a Back titration is a

non-specific method that does not differentiate starch acetate from

starch tartrate, resulting in an important overestimation of the

Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR)

were used to obtain mechanistic understanding of the acetylation of pea

over-lapping of the FTIR spectra of the native (NPS) and acetylated (APS) pea

the characteristic bands of polysaccharides, such as a broad band,

stretching vibration of C–H groups, and a series of bands in the

corresponding to the coupling mode of the C–O and C–C stretching, C-

contained new bands characteristic of the acetate groups Specifically, a

appearance of these spectral bands, together with a reduction of the

intensity of the band a due to -OH groups located along the

anhy-droglucose units, confirms the formation of starch acetates, even at the shortest reaction time (0.5 h) The intensity of these bands increased together with the reaction time However, no increase in intensity was observed after 2 h, apparently indicating that no further esterification was achieved It must be noted, however, that due to the ability of

tar-taric acid catalyst to form esters with starch, bands b and d (vibration of

ester groups) could also be dependent on the formation of tartaryl side group, and not only alkanoyl groups from acetic anhydride Similar to back titration, FTIR was not able to distinguish carboxylic acid species and related side groups Therefore, starch esters were also studied by NMR to specifically distinguish acetate from tartrate starch

NPS and APS were firstly studied by Proton Nuclear Magnetic

high DS since it presented poor solubility in DMSO and other organic

Fig 1 Degree of substitution (DStotal) of acetylated pea starch (APS) at increasing temperature and reaction times, together with the DStotal values obtained of acetylated maize starch (AMS) at 135 ◦C and increasing reaction times DStotal was determined by back titration and reported in Table 1

acid, i.e., tartaric acid catalyst, could be occurring during the process, as

at least 30 min until complete dissolution Reference signals of the

protons from the anhydroglucose units were observed between 4.4 and

linkage, whereas the small signal at 4.86 ppm corresponds to the

anomeric proton of the (α-1,6) linkage The protons of the C2 to C6

ppm due to the methyl protons of the acyl groups, demonstrating a

et al., 2017) The degree of acetylation calculated using Eq (3),

increased over time reaching the maximum after 3 h of reaction

(Table 1) Values were significantly lower than those obtained by the

classical titration, confirming the overestimation of the titration method

demonstrated to be a suitable and more accurate alternative than the

titration due to the specificity of the determination However, the poor

solubility of the starch acetates, and the possible overlapping of some

signals with the residual solvent or water, challenges the accurate

due to the C1-C6 anhydroglucose units: signal 2 at 57.6 ppm

corre-sponding to C6, signal 3 at 68.3 ppm attributed to C2, C3 and C5, signal 4

(Nielsen et al., 2018) In acetylated pea starch, two additional signals

appeared, namely signal 1 at 16.4 ppm and signal 6 at 166.6 ppm,

corresponding to the carbon of the methyl protons of the acyl group

increased with the reaction time, concomitant with a significant

underestimated the total degree of substitution (due to the

0.99) was observed between the liquid and solid-state NMR results At short reaction times (0.5 h), no reaction of the catalyst tartaric acid with the starch was observed and thus, there were no significant differences

with the DS value calculated by titration (p > 0.05) However, after 1 h,

the catalyst competed with the acetic anhydride, resulting in up to 16 %

data clearly confirmed that the DS by the chemical titration method is

Fig 2 a) Acetylation of pea starch mediated by the organocatalyst L-(+)-tartaric acid with the two possible products resulting from the reaction, starch acetate or

starch tartrate It must be noted tartaric acid-starch crosslinking can occur with any of the available hydroxyl groups of the anhydroglucose units b) FTIR spectra of native (NPS) and acetylated (APS) pea starch at different reaction times Band a (near 3470 cm− 1), b (near 1740 cm− 1), c (near 1367 cm− 1) and d (near 1217 cm− 1) correspond to the -OH groups, ester carbonyls, alkyl -CH3 deformation, C-O-C stretching vibrations, respectively c) Single Pulse-13C NMR spectra where signal 1 corresponds to the carbon of the alkyl group, signal 3 to the C2, 3, and 5 of starch, signal 2, 4 and 5 to the C6, C4 and C1, respectively, and signal 6 to the carbon of the ester group

accurate and reliable when measuring at low DS values in

organo-catalytic reactions; nevertheless, the interaction of tartaric acid with the

hydroxyl groups of the anhydroglucose residues at longer reaction times

led to an overestimation of the DS values This phenomenon agreed with

previous studies in which the contribution of tartaric acid in substitution

Vilaplana, 2020) Likewise, Nielsen et al determined the degree of

and reported overestimations between 38 and 91 % of the DS value by

derivati-zation of pea starch was mainly ruled by acetylation (from 100 % at ≤1 h

reaction times to 84 % at reactions times up to 4 h) and not by tartrate

formation The low reactivity of tartaric acid, and the efficient

acetyla-tion process of pea starch, were likely the result of several factors, such

as the type of acyl donor used (anhydride instead of acid), the low water

content of the starch sample due to the drying step prior to the reaction,

& Vilaplana, 2020) It is worth noting that, due to the dihydroxy

dicarboxylic nature of tartaric acid, a possible formation of diacetyl

tartaric acid anhydride and further esterification of the acyl groups with

dihy-droxy dicarboxylic nature of tartaric acid could likely result in

cross-linked di-starch tartrate, which notably decreased its solubility in

organic solvents (Fig S1)

3.3 Effect of tartaric acid catalyzed acetylation process on starch

granular morphology, birefringence, crystallinity, and molecular weight

(M w )

Native pea starch granules exhibited oval, spherical, kidney and

irregular shape and a bimodal size distribution, although the large

polarized light exhibited the typical birefringence attributed to their

were not altered during acetylation at reaction times ≤1 h (APS-0.5 h

reported for organocatalytic esterification of maize starch at low DS

(Tupa et al., 2013) Nevertheless, acetylation at longer reaction times

resulted in a gradual increase of granular surface roughness, decrease of

birefringence, and the appearance of granular aggregation At 3–4 h

in-crease in surface roughness and a complete loss birefringence were

detected Increase of roughness upon acylation was also observed in the

of starch with synthetic polymers as this could improve interfacial adhesion (Imre & Vilaplana, 2020)

Even in the absence of water, NPS showed a gradual loss of bire-fringence at increasing reaction times, indicating a continuous break- down of the crystalline structure during acylation X-ray diffraction patterns revealed the characteristic C-type polymorphism with peaks at diffraction angles 2θ of 15.1◦, 17.1◦, 18◦, and 23◦(Fig 3b) On one hand, organocatalytic acetylation did not alter the number and position of the diffraction peaks On the other hand, it resulted in a progressive loss of the intensity of all peaks, which followed a similar trend to that of birefringence and evidenced a continuous breakdown of the crystalline structure during acetylation This occurrence has also been reported for tartaric acid catalyzed maize starch using acetic anhydride as acyl donor, which was attributed to the introduction of acyl groups during

Vilaplana, 2020) Interestingly, even high-DS anhydride-treated samples (DSacyl ~ 3) retained their distinct granular structure (see APS-3 h in

Fig 3a) This event could be the consequence of the formation of

(Table 1), which would result in a cross-linked external shell that

The SEC-RI elution profile of NPS displayed two distinct peaks for amylose and amylopectin molecules separated at ~14 mL elution vol-ume, both represented by a single peak in the MALS detector that cor-responded to a weight average molar mass for amylopectin molecules of

with the sample concentration and injection volume used in this study and amylose molecules being highly polydisperse, they do not exhibit

shift of the peaks towards higher elution volumes, confirming hydrolytic

Similar findings were already reported before for maize starch esterified with acetic anhydride, which was explained by the effect of high

also decreased the area under the peaks and resulted in a monomodal size distribution, which could result from the coelution of amylose and amylopectin fragments and the fact that low molar mass fractions might

be particularly hydrolyzed and washed out of the sample during the

Fig 3 a) Visual appearance and morphology of native (NPS) and acetylated pea starch (APS) granules under the light (upper) and polarized light (lower)

mi-croscopy Acetylation reaction temperature was 135 ◦C and the granules were obtained after different reaction times that resulted in different DS From left to right: NPS, APS-0.5 h (DSacyl =0.39), APS-1 h (DSacyl =1.00), APS-2 h (DSacyl =2.23), APS-3 h (DSacyl =2.80), APS-4 h (DSacyl =2.63) b) X-Ray diffraction patterns of NPS and APS at different reaction times

purification Interestingly, the rate of molar mass decrease with time

was significantly lower at longer reaction times, and even the weight

esterification formed crosslinked di-starch tartrate due to the dihydroxy

dicarboxylic nature of tartaric acid

3.4 Hydrophilicity and thermal stability of tartaric acid catalyzed pea

starch acetates

Starch hydrophilicity, one of its main shortcomings for many

appli-cations as biomaterial, was indirectly investigated from the first weight

loss shown in the TGA weight-loss and derivative mass loss curves

(Fig 4b) Native pea starch depicted a first weight loss of ~10 %

2016; Tupa et al., 2013) Interestingly, this loss was indirectly correlated

hydro-philicity was significantly reduced by acetylation

The decomposition temperature of starch samples, and hence their

thermal stability and processability, increased with the degree of

acet-ylation Native pea starch showed a single weight loss peak at a

concomitant dissipation of the lower degradation step This

phenome-non was also observed to occur during the tartaric acid catalyzed

2020; Tupa et al., 2013) The first step corresponds to the condensation

of the remaining non-esterified -OH groups, whereas the second peak

was attributed to the release of acetic acid from anhydroglucose units (Imre & Vilaplana, 2020; Thiebaud et al., 1997; Tupa et al., 2013) Results showed that tartaric acid catalyzed acetylated pea starch of

DSacyl ≥2.6, whose lower temperature degradation step was completely eliminated, presented an enhanced thermal stability despite their molar mass decrease and loss of crystalline structure The substitution of the hydroxyl by alkanoyl groups in the modified starch seem to avoid inter- and intramolecular dehydration reactions ruling the decomposition of pea starch

We also investigated the effect of acetylation on the glass transition

starch granules acts as physical cross-linking avoiding the mobility of

upon acetylation was expected as a consequence of the gradual decrease

of crystallinity (Fig 3b) and molar mass (Fig 4a) However, Tg gradually

Spe-cifically, tartaryl esterification would reduce the molecular mobility, either through strong hydrogen bonding of tartaric acid with other

by the formation of covalent crosslinked di-starch tartrate, as suggested

Overall, results demonstrated that tartaric acid catalyzed acetylation

of pea starch with acetic anhydride significantly increased its

temperatures

Fig 4 a) SEC elution profiles of native (NPS) and acetylated (APS) pea starch at increasing reaction times (0.5 h to 4 h) and constant reaction temperature of 135 ◦C obtained by HPSEC-MALS-RI b) Thermal stability represented as weight loss (%) as a function of temperature (upper chart), as well as the derivative mass loss (lower chart), determined by TGA c) Thermograms showing the glass transition temperature (Tg) measured by DSC APS-0.5 h (DSacyl =0.39), APS-1 h (DSacyl =1.00), APS-

2 h (DSacyl =2.23), APS-3 h (DSacyl =2.80), APS-4 h (DSacyl =2.63)

3.5 Film-forming properties of tartaric acid catalyzed APS

Since tartaric acid catalyzed acetylation of pea starch significantly

enhanced its moisture resistance and thermal stability, we investigated

the role of an increasing degree of substitution on the film-forming

properties of APS Firstly, freestanding biofilms with APS as the only

matrix polymer were made by solvent casting Nonetheless, cohesive

films were not attained even using the acetylated pea starch with the

prepared instead, which allowed us to understand the effect of DS on

film properties, and the compatibility of APS with its native counterpart,

NPS NPS films were clear, transparent, and presented a smooth surface

(Fig 5a) When adding acetylated starch at DSacyl ≤0.4, films were still

transparent and smooth, although certain evidence for phase separation

was already visible Biofilms became gradually and visibly rougher,

darker in color, less transparent and thicker as APS with higher DS was

the substitution of -OH groups with hydrophobic monofunctional

re-agents would not only decrease inter- and intra-molecular interactions

within the polysaccharide phase, but it could also lead to less adhesion

at the interface between the composite components This could result in

meaningful phase separation and deterioration in APS film properties

(Imre et al., 2019) In fact, sharp edges and large cavities were observed

around the starch granules in the films made with APS at high DS

(Fig S4) Secondly, the decrease of molar mass during acetylation could

have lowered the availability of chains for matrix interaction Likewise,

a darker color was expected due to the dark color that results from

film thickness evidenced the uniformity of the films These values agree

with the thickness of other biofilms made with acetylated barley and rice

starches (Colussi et al., 2017; El Halal et al., 2017)

3.6 Thermal and mechanical properties and Water Vapor Permeability

The thermal decomposition of the biofilms studied by TGA revealed four degradation steps attributed to the main components of the biofilms (Fig 6a) Biofilms showed a first peak corresponding to the remaining

The weight loss % was ~6 % in NPS biofilms and a gradual loss decrease occurred when using intermediate [BAPS-0.5 h (~4.7 %)] and highly substituted APS [BAPS-1 h to -4 h (~4.1 to 4.5 %)] This event evidenced the enhanced moisture resistivity of APS biofilms compared to its NPS counterpart The first decomposition step of APS films was detected in

(Table 3) Since the decomposition of the glycerol plasticizer occurs

degra-dation step corresponds to the degradegra-dation of glycerol that is poorly interacting with starch in APS-based films Specifically, the mass loss at this temperature increased with the degree of substitution A second

which was attributed to the decomposition of native starch Logically, the area of this step decreased as DS increased, since technically there is less non-esterified starch present in the films No major differences were

by all likelihood represents the decomposition of alkanoyl groups in the

limits intramolecular dehydration of polysaccharides by reducing the

turn, delays the initiation of thermal decomposition

The mechanical properties of APS-films were measured under ten-sion, and the tensile strength (MPa), elongation at break (%), and Young's Modulus (MPa) were determined from the stress-strain curves of

to native starch films The reduction of intermolecular interactions within the starch phase as a consequence of surface lamellar acetylation

in low DS starch could explain the alteration of the blend morphology and decreased particle size (as observed in Fig S4) We believe that this occurrence could lead to a more homogeneous dispersion of the dispersed phase It is noteworthy that at short reaction time, APS

than that reported for tartaric acid catalyzed maize starch using acetic

could have played a minor negative role in APS-0.5 films Nevertheless, APS with higher DS showed a gradual decrease in the tensile strength

Table 2

Glass transition temperature (Tg) and thermal degradation (Td) of native (NPS)

and acetylated pea starch (APS) studied by DSC and TGA, respectively

NPS APS-0.5

h APS-1 h APS-2 h APS-3 h APS-4 h

T g ( ◦ C) * 110.8

± 2.2 c 136.6

± 3.4 b 129.5

± 2.6 b 130.3

± 2.9 b 137.1

± 2.8 b 153.6

± 5.6 a

T d1 ( ◦ C) 318.9

± 0.6 a 313.7

± 0.9 b 289.2

Area T d1

(mg/ o C) 19.5 ±0.1 a 6.7 ±

0.2 b 1.7 ±

T d2 ( ◦ C) – 373.8

± 0.4 ab 374.1

± 1.3 a 369.3

± 0.4 c 374.5

± 0.7 a 372.3

± 0.1 ab Area T d2

(mg/ o C) – 2.2 ±0.1 c 9.4 ±

0.2 b 21.3 ±

0.2 a 22.9 ±

0.3 a 19.3 ±

3.9 a

*Tg, glass transition temperature measured in a second heating cycle by DSC

Td1 and Td2 represent the degradation peaks detected by TGA Values are

expressed as average (n = 2) ± standard deviations Values followed by different

letters within each parameter (row) indicate significant differences (p ≤ 0.05) -,

not detected

Fig 5 Biofilms made of (a) native pea starch, and blends of native: acetylated (3:1, w:w) pea starch of (b) APS-0.5 h (DSacyl =0.39), (c) APS-1 h (DSacyl =1.00), (d) APS-2 h (DSacyl =2.23), (e) APS-3 h (DSacyl =2.80), (f) APS-4 h (DSacyl =2.63)

governing film roughness and transparency (see Section 3.5) could also

explain mechanical properties Schmidt et al also reported improved

tensile strength at low DS values and poorer mechanical resistance at

high DS investigating NaOH catalyzed acetylated cassava starch biofilms

(Schmidt et al., 2019) The elongation at break (EB%) using APS DSacyl

≤ 1 was similar or slightly worse than that of NPS films (Fig 6b,

Table 3), in agreement with other low acetylated starch films (L´opez

et al., 2011; Schmidt et al., 2019) Nonetheless, EB% improved when

retained crystallinity of low acetylated starch probably makes these

Table 3

Mechanical, thermal and barrier properties of biofilms made with native pea starch (BNPS) or native/acetylated pea starch blends (BAPS) at a 3:1 ratio (w/w)

Mechanical properties

Thickness (mm) 0.09 ± 0.00 c 0.08 ± 0.00 c 0.10 ± 0.00 b 0.11 ± 0.00 a 0.11 ± 0.02 ab 0.12 ± 0.01 a Tensile Strength (MPa) 17.8 ± 1.3 b 20.1 ± 1.7 a 13.9 ± 0.8 c 7.1 ± 0.4 d 6.4 ± 0.9 d 5.8 ± 0.3 d Elongation at break (%) 4.3 ± 0.1 ab 2.4 ± 0.7 b 3.1 ± 0.3 b 5.4 ± 0.7 a 5.6 ± 0.5 a 3.8 ± 0.5 b Elastic modulus (MPa) 804.9 ± 14.4 b 1003.5 ± 136.2 a 689.3 ± 83.0 c 332.5 ± 43.6 d 263.6 ± 25.7 d 302.7 ± 10.8 d

Thermal stability (TGA)

T d2 ( ◦ C) 320.6 ± 2.9 b 323.1 ± 0.1 ab 323.5 ± 0.5 ab 324.1 ± 0.4 a 324.2 ± 0.5 a 322.2 ± 0.7 ab Area T d2 (mg/ o C) 17.6 ± 2.2 a 11.6 ± 0.3 b 11.0 ± 0.2 b 10.1 ± 0.7 b 9.6 ± 0.0 b 10.7 ± 0.4 b

Water Vapor Permeability (WVP)

WVP (g⋅mm)/(m 2 ⋅day⋅KPa) 1.03 ± 0.07 ab 0.74 ± 0.09 c 0.92 ± 0.00 b 1.11 ± 0.02 a 0.93 ± 0.01 b 1.02 ± 0.08 ab Values followed by different letters within each parameter (row) indicate significant differences (p ≤ 0.05) Td1, Td2, and Td3 represent the degradation temperature peaks detected by TGA -, not detected BAPS-0.5 h (DSacyl =0.39), BAPS-1 h (DSacyl =1.00), BAPS-2 h (DSacyl =2.23), BAPS-3 h (DSacyl =2.80), BAPS-4 h (DSacyl = 2.63)

Fig 6 a) Thermal stability of native (BNPS) and acetylated pea starch (BAPS) biofilms, made with acetylated pea starch at different DS, represented as weight loss

(%) as a function of temperature (left), as well as the derivative mass loss (right), determined by TGA b) Strain stress curve of BNPS and BAPS biofilms APS-0.5 h (DSacyl =0.39), APS-1 h (DSacyl =1.00), APS-2 h (DSacyl =2.23), APS-3 h (DSacyl =2.80), APS-4 h (DSacyl =2.63)