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Official URL: https://doi.org/10.1007/s00284-019-01804-7

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Choque, Élodie and Durrieu, Vanessa and Alric, Isabelle and Raynal,
José and Mathieu, Florence Impact of Spray-Drying on Biological
Properties of Chitosan Matrices Supplemented with Antioxidant Fungal
Extracts for Wine Applications. (2019) Current Microbiology, 77 (2). 210-219.
ISSN 0343-8651

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https://doi.org/10.1007/s00284-019-01804-7

Impact of Spray‑Drying on Biological Properties of Chitosan Matrices

Supplemented with Antioxidant Fungal Extracts for Wine Applications
Elodie Choque1,2 · Vanessa Durrieu3 · Isabelle Alric3 · José Raynal1 · Florence Mathieu1

Abstract
Black aspergilli produce many bioactive compounds: enzymes, organic acids, and secondary metabolites. One such fungus,
Aspergillus tubingensis G131, isolated from French Mediterranean vineyards, produces secondary metabolites with anti-
oxidant properties that can be extracted with ethanol. In this study, crude antioxidant extracts obtained from A. tubingensis
G131 cultures were encapsulated with two types of chitosan matrix. Spray-drying was used to obtain dried particles from a
dispersion of fungal crude extracts in a solution of the coating agent chitosan. This process appeared to be an efficient method
for obtaining a dry extract with antioxidant activity. Three types of fungal extracts, with different antioxidant capacities,
were produced: two different concentrations of crude extract and a semi-purified extract. In this study, the chitosan matrices
for encapsulation were chosen on the basis of their antimicrobial activities for wine applications. Classical low molecular
weight chitosan was compared with NoBrett Inside® which is already used to prevent the development of Brettanomyces
spp. in wine. The objective of this study was to confirm that both antioxidant (fungal extract) and antimicrobial (chitosan)
properties were preserved after spray-drying. The combination of these two properties and the powder formulation of this
entirely natural product would make it a good alternative to chemicals, such as sulfites, in the food and wine industries.

Keywords Fungal metabolites · Chitosan · Microencapsulation · Spray-drying · Antioxidant

Abbreviations SEM  Scanning electron microscopy

ABTS 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic TEAC Trolox equivalent antioxidant capacity
Acid
AE  Additive effect
AN  Antagonistic effect Introduction
CE  Crude extract
LMW  Low molecular weight One of the key challenges in the wine industry is con-
NBI  No Brett ­inside® trolling wine stability during production, aging, and after
NGPs Naphtho-gamma-pyrones bottling. During wine aging, the principal objectives of
PE  Semi-purified extract the winemaker are preventing wine spoilage and stabiliz-
RT Room temperature ing wine color by limiting oxidation. Sulfites are added at
SPE  Solid-phase extraction various steps in the winemaking process to achieve these
SE  Synergistic effect objectives. However, it has been suggested that sulfites
may have adverse effects on human health, such as pseu-
doallergies, and winemakers are therefore now trying to
* Vanessa Durrieu
vanessa.durrieu@ensiacet.fr limit the use of sulfites in the winemaking process [1–3].
Indeed, wine consumers prefer high-quality less-processed
1
Laboratoire de Génie Chimique, Université de Toulouse, wines [4, 5]. For winemakers to meet this demand, the
CNRS, INPT, UPS, Toulouse, France development of new preservative agents or stabilization
2
Unité de Recherche Biologie Des Plantes Et Innovation techniques is required. Natural products are currently
(BIOPI‑EA 3900), Université de Picardie Jules Verne, 33 rue defined as metabolites produced by living organisms and/
Saint Leu, 80039 Amiens Cedex, France
or naturally occurring in nature. Such metabolites from
3
Laboratoire de Chimie Agro‑Industrielle (LCA), Université various organisms are capable of preventing microbial
de Toulouse, INRA, INPT, Toulouse, France
spoilage, and thus are a suitable alternative to the use convert liquid preparations into powders, or to improve their
of synthetic products [6]. Among them, chitosan meets dispersibility in water [20].
these requirements, and the European Union (EU) and Various microencapsulation technologies, such as spray-
International Organization of Vine and Wine (OIV) have drying, prilling, coacervation, extrusion, and in situ polym-
already approved its use for wine applications. The OIV/ erization, are available. Spray-drying is one of the simplest
OENO 338A/2009 (International Organization of Vine and and most widely used processes due to its relatively low cost
Wine (OIV) 2009) resolution added the use of chitosan and efficiency and the availability of appropriate industrial
in winemaking to the International Code of Oenological equipment [21]. Antioxidant encapsulation by the spray-
Practice, specifying that the maximum dose of chitosan drying method has already been described for various wall
used to reduce ochratoxin A (OTA) levels must not exceed materials, such as plant proteins [22–24], milk proteins [25],
500 g/hL. Chitosan is a linear polysaccharide composed of various carbohydrates, including maltodextrins [26–30], and
two repeating units (d-glucosamine (GlcN) and N-acetyl- blends of maltodextrin/gum arabic [31], or maltodextrin/K-
d-glucosamine (GLcNAc)) randomly distributed along the carrageenan [32], and for chitosan [33–35].
polymer chain and linked by β(1-4)-bonds. Various activi- The objectives of this work were to study the antimicro-
ties have been demonstrated for different types of chitosan: bial and antioxidant capacities of chitosan/fungal extract
as a fining and protein stabilizing agent, a preservative and microparticles obtained by spray-drying for potential appli-
an antimicrobial agent [3, 7, 8]. Chitosan has antimicrobial cations in the food industry, including the maintenance of
effects against lactic acid bacteria, acetic acid bacteria, wine stability. Indeed, the combination of these two types
fungi, and undesirable yeasts, such as Brettanomyces sp., of activity should prevent both the spoilage and oxidation
during aging, but it is permissive for the growth of Sac- of the wine. Microparticles of this type will not only com-
charomyces species [7, 9–12]. bine two biological properties, but also will incorporate into
For wine aging applications, it would be useful to cou- food in solid form, without dissolution, thereby preventing
ple the antimicrobial properties of chitosan with antioxi- alterations to organoleptic qualities, such as color [35]. The
dant activity to stabilize wine color. In this study, an ethanol chitosan/fungal extract microparticles were subjected to
extract of Aspergillus tubingensis G131 was chosen. This physicochemical (particle size, morphology and moisture)
black Aspergilli strain was isolated during a survey on the and biological (impact on yeasts growth, antioxidant capac-
occurrence of OTA producers on grapes from various French ity) characterization.
vineyards [13]. A. tubingensis G131, isolated from a French
Mediterranean Vineyard, does not produce OTA [14]. An
ethanol extract of a sporulated mycelial cake from this strain Materials and Methods
was found to be dark gold in color, due to the presence of
melanin, and has interesting antioxidant properties. Equiva- Chemicals
lent extracts from other black Aspergilli strains have already
been reported to have antioxidant activity. For example, a Chitosan 1 (NBI)
methanol extract from an A. niger strain was found to have
beneficial effects on rat growth and hepatoprotective activity This chitosan preparation was a powder with particles of less
[15]. Another organic extract of A. niger was shown to pre- than 50 µm in diameter, and a product of the deacetylation of
vent lard oxidation [16]. These findings suggest that organic chitin extracted from A. niger supplied by KitoZyme com-
extracts of non-mycotoxigenic black Aspergilli are of poten- pany (Herstal, Belgium): KiOfine ­B® or No Brett ­Inside®
tial interest for their use as preservatives in the food industry. (commercially available products). This product was less
A suitable strategy for efficiently combining the antimi- than 30% acetylated and the viscosity of a 1% solution in
crobial properties of chitosan with the antioxidant activity acetic acid was about 4 mPa.s.
of the chosen fungal extract was therefore required. Micro-
encapsulation appeared to be an effective approach. Chitosan 2 (LMW)
Interest in antioxidant encapsulation has recently
increased, particularly in the food industry. This process The chitosan preparation was obtained from Glentham Life
makes it possible to increase the stability of bioactive com- Sciences (Wiltshire, United Kingdom) and used without
pounds during processing and storage and prevents undesir- purification. It had a very low molecular weight (average
able interactions with the other components of a formulation molecular weight: 30,000 g mol−1) and was less than 10%
[17–19]. The protective mechanism of microencapsulation acetylated.
involves the formation of a membrane wall to enclose drop- These two chitosans had low molecular weights and
lets or particles of the encapsulated ingredient, thereby pro- acetylation levels, features known to be associated with the
tecting it, improving its stability, and making it possible to antibacterial activity of chitosan [36].
 The anhydrous ethanol (Fisher®), acetic acid, and other Estimation of the Dry Weight of Fungal Extracts
chemicals (Sigma-Aldrich®) used were of analytical grade.
The dry weight of all the fungal extracts obtained was
determined with an EM120-HR Moisture Analyzer (Pre-
Fungal Extracts cisa Gravimetrics AG, Dietikon, Switzerland) by drying to
constant weight at 70 °C.
Production of the Crude Fungal Extract

Czapek Yeast Broth (CYB—30 g/L Saccharose, 5 g/L Yeast HPLC Characterisation of the Fungal Extracts
Extract, 2 g/L NaNO3, 0.25 g/L KCl, 0.25 g/L MgSO4.7H2O,
0.005 g/L FeSO 4.7H 2O, 0.5 g/L K 2HPO 4, 0.001 g/L HPLC analyses were performed as described in Choque
ZnSO4.7H2O and 0.0005 g/L CuSO4.7H2O) was inoculated et al. [14].
with 107 spores of A. tubingensis G131, and the resulting
fungal cultures were incubated for 7 days at 30 °C. The
mycelial cake was then separated from the culture medium. Microencapsulation Methodology
The mycelial cake only was then covered with anhydrous
ethanol. The mycelium/ethanol mixture was incubated for Preparation of the Solution
20 min at room temperature (RT) and then subjected to soni-
cation for 20 min at 50 Hz. The sonicated mycelium/ethanol Solution of 1% (w/w) chitosan in 1% (v/v) aqueous ace-
mixture was filtered once through 113 V grade Whatman tic acid was prepared with mechanical stirring (500 rpm)
filter paper. The filtered extract obtained was stored in the at RT. Fungal extracts in ethanol (CE*3, CE*6, and PE*2)
dark at 4 °C until required, and is referred here as the crude were then added to the chitosan solution, 80% of chitosan
extract (CE). solution and 20% of fungal extract, with mechanical stirring
(500 rpm) to mix them before the spray-drying process.

Concentration by Rotary Evaporation

Preparation of Spray‑Dried Microparticles
The crude extract was concentrated (volume to volume) with
a rotary evaporator. The conditions for evaporation were Obtained chitosan/fungal extract solutions were spray-dried
as follows : 60 °C, rotation at 150 rpm, and uncontrolled in a Mini Spray Dryer B-290 (Büchi, Flawil, Switzerland) in
vacuum and cooling with distilled tap water at 15 °C. Crude the open mode under the following conditions: inlet air tem-
extracts (CEs) were named on the basis of their concentra- perature at 120 ± 4 °C and outlet temperature at 75 ± 4 °C,
tion yield. In this study, two concentrated extracts were used: drying air flow rate of 470 L/h, liquid feed flow rate varying
CE*3 (3 V/V: 3 volumes were evaporated in 1 remaining from 0.33 to 0.48 L/h and 100% aspiration. Microparticles
volume) and CE*6 (6 V/V: 6 volumes were evaporated in 1 were collected from the container, sealed hermetically in an
remaining volume). opaque packaging, and stored at room temperature.
Spray-drying yield was calculated as follows:
Spray − drying yield (%) = MP ∕MRM × 100
Solid‑Phase Extraction
where MP is the dry mass of the microparticles collected and
Solid-phase extraction (SPE) was performed on a C18 MRM is the initial dry mass of the solids in the solution of
Hypersep column (Thermo Fischer Scientific)—Bed weight: chitosan and fungal extract.
5 g; Sorbent: Hypersep C18; Particle Size: 40–60 μm; and
column capacity: 25 mL. Columns were equilibrated with
Physicochemical Characterization
five volumes of acetonitrile and three volumes of MilliQ
of Microencapsulated Matrices
water. 5 mL of CE*3 was deposited on the column and
washed with two volumes of water/acetonitrile (70:30 mix-
Moisture Content
ture). Finally, a semi-purified fraction was eluted in one vol-
ume of anhydrous ethanol. This semi-purified SPE product
The moisture content of all the powders obtained was deter-
was then concentrated by rotary evaporation (2 V/V: 2 vol-
mined with an EM120-HR Moisture Analyzer (Precisa
umes were evaporated in 1 remaining volume) and named
Gravimetrics AG, Dietikon, Swiss) by drying to constant
(PE*2).
weight at 105 °C.
Microparticles Size Distribution were added 16 h before inoculation, and the medium was
allowed to equilibrate at 20 °C. The YEPD medium was then
The particle size distribution of dried microparticles was inoculated to an O­ D600nm of 0.1 with yeast preculture. The
determined from the scattering pattern of the transverse growth of the yeasts was monitored by spectrophotometry
laser light with a Scirocco 2000 instrument (Malvern Instru- at λ = 600 nm,at times 0, 8 h, 24 h, and 48 h. OD was also
ments, Worcestershire, UK). Mean particle diameter ranged estimated before inoculation to ensure the correct estimation
from 0.2 to 2000 µm. The following parameters were used: of yeast growth. The experiments were carried out in dupli-
refractive index of 1.52, pressure of air of dispersion of 4 cate, at 20 °C, and flasks were stirred just after inoculation
bars, degree of vibration of 70%. The volume-based particle and immediately before sampling.
diameter ­(D43 or ­Dv) was calculated as the mean of three
measurements per sample. Trolox Equivalent Antioxidant Capacity

Microparticle Microstructure The antioxidant activity of the fungal extracts was deter-
mined in the TEAC test, according to a protocol adapted
The morphology of microparticles was examined by scan- from a previous study [37]. ­ABTS+ was produced by the
ning electron microscopy (SEM). The particles were depos- reaction of 7 mM of ABTS (2,2′-azino-bis(3-ethylben-
ited on conductive double-faced adhesive tape and sputter- zothiazoline-6-sulfonic acid)) (Sigma-Aldrich, > 99%
coated with silver. The microparticles were frozen in liquid HPLC grade) with 2.5 mM of ­K2S2O8 (sodium persulfate)
nitrogen and broken up in a mortar, for examination of their (Sigma-Aldrich, > 99% ACS reagent) in distilled water for
internal structure. SEM observations were performed with 16 h at RT, in the dark. The concentration of A ­ BTS+ was
a LEO435VP scanning electron microscope (LEO Electron then adjusted by dilution to an absorbance of 0.7 ± 0.02
Microscopy Ltd., Cambridge, UK) operating at 8 kV. (at 734 nm) on a spectrophotometer (Anthelie advanced,
SECONAM). A stock solution at 150 mM was prepared for
Microencapsulated Matrix Suspension Trolox (Sigma-Aldrich). A 100 μL aliquot of the desired
sample, at the appropriate dilution, or the Trolox standard
Each microencapsulated matrix was dissolved in acidified (final concentration of 2.5, 5, 10 or 15 μM) was added to
MilliQ water (pH 3.0, acetic acid), 12% ethanol (pH3.0, 900 μLl of the diluted ­ABTS+. Absorbance was measured at
acetic acid) or 100% ethanol to give a 3 g/L (dry weight) 734 nm after incubation in the dark, at RT, for 6 min. Each
solution. The weighted powder was incubated in the appro- sample was tested three times in duplicate. TEAC value was
priate solvent for 24 h at RT and then stored at 4 °C. Each estimated in mM as described by Re et al. [37].
experiment was run in triplicate.
Classification of Additive, Synergistic
Effect of Microencapsulated Matrices on Enological
or Antagonistic Effects
Yeasts Growth
As described by Ribeiro et al. [38], theoretical values for
The effects of spray-drying chitosan and its combina-
the antioxidant activity of the assayed extract mixture were
tion with a fungal antioxidant on growth of two enologi-
calculated as the weighted mean of the experimentally deter-
cal strains—Saccharomyces cerevisiae and Brettanomyces
mined TEAC values of the individual extracts. For example,
bruxellensis—were assessed.
for CE*3, the estimated TEAC was based on the dilution
Yeasts were precultured in YEPD (10 g/L yeast extract,
factor applied to both the chitosan and fungal extracts in
20 g/L meat peptone, 20 g/L d-glucose) for 16 h at 30 °C.
the microencapsulated matrix suspension. It was therefore
Two concentrations of chitosan were used, based on the
calculated as follows:
results of Taillandier et al. (Taillandier et al., 2015), describ-
ing the mode of action of NBI chitosan on B. bruxellensis
( ( ))
TEACest = TEACfungal extract × Mfe− c ∕ Mfe− i
inhibition: 40 mg/L and 400 mg/L [11]. These concentra- ( ( ))
+ TEACchitosan × Mc− c ∕ Mc− i
tions were chosen as they correspond to the ends of the
standard range of NBI showing an action on B. bruxellensis. where Mfe-c is the dry mass of fungal extract in the micro-
The controls were culture media without any supplementa- encapsulated matrix suspension; Mfe-i is the dry mass of the
tion, and culture media supplemented with low molecular initial fungal extract; Mc-c is the dry mass of chitosan in the
weight chitosan or with No Brett Inside chitosan that had microencapsulated matrix suspension; and Mc-i is the dry
not been spray-dried. The supplements to the YEPD medium mass of the initial chitosan solution.
 Effects were classified as additive (AE), synergistic (SE) preventing obstruction of the nozzle during the spray-drying
or antagonistic (AN) as described by Ribeiro et al. [38]. process and increasing encapsulation efficiency [21, 40].
AE: theoretical and experimental values differed by less than For the spray-drying parameters, the chosen inlet tem-
5%. SE: experimental values were at least 5% higher than perature of 120 °C was sufficient to evaporate both sol-
theoretical values. AN: experimental values were at least vents and acetic acid (which has a boiling point is 118 °C),
5% lower than theoretical values. Lower TEAC values are and the feed spray rate was adapted to maintain the out-
associated with lower levels of antioxidant activity. let temperature around 75 °C, ensuring that the particle
dried correctly with no alteration of the properties of the
Statistical Analyses extracts. Appropriate adjustment between the inlet temper-
ature and the feed flow is essential to ensure the evapora-
Data are presented as means ± SD for triplicate experiments. tion of maximum of the liquid sprayed before the droplets
Statistical analyses were performed with jamovi (jamovi meet the drying chamber walls, to optimize the production
project (2017), jamovi (Version 0.8)). One-way and two-way yield [40].
ANOVA tests were used to assess the significance of differ- The incorporation of ethanol extract, even with a high
ences for each variable (p < 0.05). Tukey post hoc test was proportion of fungal material (PE*2: 1.39 ± 0.03 g/L; CE*3:
applied to determine the significance of differences between 29.4 ± 0.7 g/L and CE*6: 55.1 ± 1.2 g/L (dry weight)),
conditions. made it possible to obtain production yields of around 80%
(Table 1). These results were highly satisfactory and signifi-
cantly better than published production yields for the use of
Results chitosan as the encapsulation material, which vary from 23
to 73%, and are generally around 50% [21, 41].
Microencapsulation of Fungal Extracts Mean particle size ranged from 1.17 to 4.35 µm, consist-
ent with published values for chitosan and loaded chitosan
Spray-drying was used to prepare chitosan and ethanol microparticles, which are generally between 1 and 5 µm
extract-loaded chitosan microparticles. Several parameters [21]. The results of the statistical analysis suggested that
can affect the characteristics of the obtained microspheres, mean particle size depends on the type of fungal extract used
including polymer concentration, solvent and assay condi- for supplementation, but not of the type of chitosan. These
tions (inlet temperature, spray rate of feed, etc.) [39]. values were confirmed by SEM observations (Fig. 1). This
For these experiments, chitosan concentration was fixed could be linked to the composition of the extract. HPLC
at 1% (w/w) in 1% (v/v) aqueous acetic acid to ensure com- analysis of the extract were performed as described in
plete dissolution of the chitosan and solutions with a suit- Choque et al. [14]. Results show that fungal extracts are
able viscosity for spraying. Ethanol extracts (CE*3, CE*6, composed of two main compounds: melanin (black pig-
PE*2, 100 mL in each case) were then added to give an ment), and Naphtho-Gamma-Pyrones (NGPs). The propor-
80/20 water/ethanol ratio, which maintained suitable levels tion of NGPs of each extract was calculated with chromato-
of solubility of both chitosan and the extracts in the solution, gram analysis. The proportions were 94.4 ± 2.3% of NGPs

Table 1  Spray-drying yield and Chitosan Fungal extract Initial dry Spray-drying Mean microparti- Particle size coef-
mean micro particle diameter weight (g) yield (%) cle size (μm) ficient of variation
(%)

LMW None 3.998 61 1.52 ± 0.03a 1.64

PE*2 4.100 81 2.49 ± 0.01b 0.40
CE*3 6.944 80 3.29 ± 0.04c 1.09
CE*6 9.497 81 4.35 ± 0.33d 7.59
NBI None 4.054 78 1.17 ± 0.05a 4.10
PE*2 4.170 74 2.26 ± 0.01b 0.35
CE*3 6.961 80 3.13 ± 0.01c 0.10
CE*6 9.492 79 3.37 ± 0.14c 4.24

LMW low molecular weight chitosan, NBI No Brett ­Inside® chitosan; None anhydrous ethanol control,
PE*2 SPE semi-purified fungal extract, CE*3 fungal extract concentrated by rotary evaporation (3 V/V),
CE*6 fungal extract concentrated by rotary evaporation (6 V/V)
Data with the same letter are not significantly different (two-way ANOVA with Tukey post hoc correction,
p-value < 0.05)
Fig. 1  Scanning electron micrographs of LMW + CE*6 microparticles (a), NBI + CE*6 microparticles (b), and NBI + None microparticles (c)

in PE*2; 34.2 ± 2.5% of NGPs in CE*3, and 28.3 ± 1.8% of whereas antioxidant supplementation of the chitosan matrix
NGPs in CE*6. The more the NGPs are present, the less the considerably decreased antimicrobial efficacy, at least for
melanin is. Thus, it appears that the particle size of the chi- CE*3 and CE*6 supplementation. The addition of a too large
tosan microparticles is linked to the proportion of melanin quantity of antioxidant might, therefore, limit the antimi-
in the fungal extract. The more the melanin is present, the crobial efficacy of chitosan or strong presence of melanin in
higher is the particle size. those matrices could limit the mode of action of chitosan.
The microparticles produced were spherical and of regu-
lar shape, but with a slightly cracked surface. No signifi- Antioxidant Activity of Microencapsulated Matrices
cant difference was observed between chitosan and fungal
extracts, other than for the mean size of the microparticles Regarding the antioxidant capacity of the fungal extracts
produced. Indeed, initial dry weight differed between fungal before microencapsulation, a relation can be found between
ethanol extracts and higher initial dry weights of the spray- the quantity of NGPs present in an extract and its antioxidant
dried sample were associated with larger mean microparticle capacity. Indeed, an antioxidant capacity between 1.2- and
size. This reflects a higher concentration of matter in the 1.6-mM TEAC could be found per gram of NGPs present
sprayed droplets. in the extract (not statistically different). The antioxidant
capacity of NGPs have previously been described confirm-
Effect of Microencapsulated Matrices on the Growth ing this hypothesis [15, 16, 43].
of Enological Yeasts Three types of microencapsulated matrix suspensions
were tested: anhydrous ethanol (the solvent used to obtain
The approach used here aimed to combine the antimicro- the antioxidant fungal extract); acidified ethanol 12% (pH
bial properties of chitosan with the antioxidant properties 3) to represent conditions equivalent to those found in wine;
of the fungal extract. No Brett I­ nside® is a commercial chi- and acidified water (pH 3) to mimic grape must. Antioxi-
tosan, obtained from the fungus Aspergillus niger, which dant activity was limited in anhydrous ethanol suspensions
is already on sale for limiting wine spoilage due to Bret- (Table 2) possibly due to the very low solubility of chitosan
tanomyces bruxellensis [11]. This micro-organism is rec- in organic solvents. In such conditions, the chitosan envelope
ognized as a major source of contamination in wine and would prevent the release of the antioxidant molecules into
as the principal cause of a “horse sweat” flavor [42]. Bret- the medium, thereby limiting antioxidant activity. Alterna-
tanomyces bruxellensis can develop in both musts and wine, tively, there may be an antagonistic effect between chitosan
throughout the entire winemaking process. As described by and the antioxidant molecule of the fungal extract in this
Taillandier et al., No Brett I­ nside® chitosan (NBI) seemed type of solvent.
to be generally more effective than low molecular weight By contrast, in the suspensions simulating wine (12%
chitosan (LMW) in the winemaking context. NBI seemed ethanol) and must (acidified water), a synergistic effect on
to have less impact on the growth of S. cerevisiae, which is antioxidant properties was observed (Table 2).
required for alcoholic fermentation, and a stronger impact
on the growth of B. bruxellensis, the chief contaminant, than
LMW (Fig. 2).
Conversely, the spray-drying technique has no impact
on the slowing of B. bruxellensis development (Fig. 2b),
Fig. 2  Growth of Saccharomyces cerevisiae (a) and Brettanomy- gal extract concentrated by rotary evaporation (3 V/V), CE*6 fungal
ces bruxellensis (b) populations in the presence of different types of extract concentrated by rotary evaporation (6 V/V). Letters indicate
chitosan, as assessed by measuring ΔOD600nm. LMW low molecular values significantly different from the control (one-way ANOVA with
weight chitosan, NBI No Brett I­nside® chitosan; None anhydrous Bonferroni post hoc correction; a: p-value < 0.05; b: p-value < 0.005;
ethanol control, PE*2 SPE semi-purified fungal extract, CE*3 fun- c: p-value < 0.001)

Discussion properties) and to reduce the oxidation of grapes to pre-

serve wine color by preventing juice browning (antioxidant
This study shows that the supplementation of chitosan properties). The greatest risk in must is enzymatic brown-
matrices, already commercially available, with an antioxi- ing [44]. Alternatives to sulfites have already been proposed
dant extract, produced from a filamentous fungus isolated for preventing browning at this stage: salicylic acid during
from a Mediterranean grapevine, could extend the use of this harvesting, glutathione or patatin, and a fining agent limit-
additive to the entire winemaking process as a sulfites alter- ing browning [44–46]. In this study, an antioxidant extract
native. As discussed in the introduction, sulfites are added produced from a fungus isolated from Mediterranean grape-
during the winemaking process for two reasons: to limit vines was selected as a natural product to supplement both
the growth of undesirable microorganisms (antimicrobial LMW and NBI for use in the winemaking process. The two
Table 2  Antioxidant capacity of microencapsulated matrix suspensions at 3 g/L (dry weight)
Ethanol 100% Ethanol 12% Acidified water
TEACest (mM) TEACexp (mM) TEACest (mM) TEACexp (mM) TEACest (mM) TEACexp (mM)

Control
CE*3 11.22 ± 0.53
CE*6 18.66 ± 0.21
PE*2 2.2 ± 0.08
LMW 0.06 ± 0.001 0.22 ± 0.005 0.31 ± 0.025
NBI N.D. ± 0 0.11 ± 0.002 0.19 ± 0.005
LMW
CE*3 0.52 0.16d ± 0.017 0.61 1.15b ± 0.021 0.66 1.56 ± 0.036
CE*6 0.61 0.38c ± 0.016 0.68 0.76e ± 0.005 0.72 0.90 ± 0.018
PE*2 0.22 0.44c ± 0.01 0.37 1.09b ± 0.036 0.45 1.4a ± 0.04
NBI
CE*3 0.48 0.16d ± 0.01 0.54 1.26a ± 0.055 0.59 1.4a ± 0.07
CE*6 0.59 0.40c ± 0.009 0.63 1.13b ± 0.056 0.67 0.97b ± 0.018
PE*2 0.16 0.34c ± 0.022 0.26 0.83e ± 0.034 0.34 0.94b ± 0.004

The mean of a triplicate analysis is indicated (SD < 0.07)

LMW low molecular weight chitosan, NBI No Brett I­ nside® chitosan, PE*2 SPE semi-purified fungal extract, CE*3 fungal extract concentrated
by rotary evaporation (3 V/V), CE*6 fungal extract concentrated by rotary evaporation (6 V/V), TEACest estimated TEAC, TEACexp experimental
TEAC
Synergistic effect (SE) is indicated in bold; Antagonistic effect (AN) is underlined
Data with the same letter are not significantly different (two-way ANOVA with Tukey post hoc correction, p-value < 0.05)

natural active ingredients were combined by spray-drying was observed for the chitosan/fungal extract combina-
microencapsulation to generate a powder that is easy to use tion, highlighting the potential value of this powder for
and to store. The spray-drying technique seems to decrease the use in the winemaking process. PE*2 supplemented
the negative impact of chitosan on S. cerevisiae growth, with matrices had the strongest antioxidant properties, with a
synergistic effects, increasing the antioxidant capacity of synergistic effect of 2.8 to 3.2 times the expected value.
the mixture. Indeed, the growth of S. cerevisiae was signifi- These results are consistent with those of Sansone and
cantly slowed in the presence of the commercial chitosan at colleagues (2014), showing small particles size generally
the concentration recommended for limiting the growth of B. increases microparticle dissolution and the release of the
bruxellensis [11]. This slowing of growth has been reported encapsulated active ingredient (Sansone et al. [41]). Under
to be due to a phenomenon of yeast absorption by the poly- laboratory conditions, in simple media, the antimicrobial
mer [47]. This slower growth of S. cerevisiae may slow the and antioxidant properties were combined, revealing this
alcoholic fermentation, prolonging the winemaking process, powder to be a promising alternative to sulfites for use
if chitosan is added to the must stage. It limits the use of the in winemaking. Under the conditions tested, the PE*2-
commercial NBI product for the treatment of wines before supplemented matrix is a good candidate for use in wine
bottling. Interestingly, the effect of chitosan on S. cerevisiae applications. Indeed, the spray-drying technique limited
growth seems to be reduced by spray-drying and/or anti- the effects of chitosan on S. cerevisiae growth, whereas
oxidant supplementation. This could allow a wider use of the effect on B. bruxellensis growth was similar to that of
chitosan in winemaking processes, including its addition to NBI used in the recommended conditions in wine (Con-
the must before the initiation of alcoholic fermentation. trol, 400 mg/L). The addition of the NBI PE*2 matrix to
The experimental antioxidant capacity of most powders the must could therefore be considered in winemaking.
was two to three times higher than the estimated value However, for confirmation of this potential, scaling up of
for a combination of the antioxidant activities of chitosan the fungal antioxidant production has to be shown in order
and the chosen fungal extract [48]. The spray-drying of to manufacture the process, and recent advances have been
a combination of two antioxidant fungal extracts (Suil- made on this step [49]. Then, both properties (antioxi-
lus luteus and Coprinopsis atramentaria) has already dant and antimicrobial) will need to be tested directly on a
been shown to double the antioxidant activity over that must, with evaluations of browning and microbial spoilage
expected (Ribeiro et al. [38]). A similar synergistic effect throughout the winemaking process and assessments of the
release of the active ingredients from the powder into a A-producing fungi in French grapes and characterization of new
complex medium such as must or wine. Finally, innocuity naphtho-gamma-pyrone polyketide (aurasperone G) isolated from
Aspergillus niger C-433. J Agric Food Chem 55:8920–8927
of the product should be confirmed. However, previous 14. Choque E, Klopp C, Valiere S, Raynal J, Mathieu F (2018) Whole-
studies have already shown beneficial effect on rats’ health genome sequencing of Aspergillus tubingensis G131 and overview
with a daily intake of NGPs [11, 50]. of its secondary metabolism potential. BMC Genomics 19:200
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EC realizes the crude extract production, extract purification, antioxi- gillus niger. J Sci Food Agric 26:1357–1369
dant, and antimicrobial analyses. VD and IA realize microencapsula- 17. Mozafari MR, Flanagan J, Matia-Merino L, Awati A, Omri A,
tion, physicochemical properties of the obtained particles, and scanning Suntres ZE, Singh H (2006) Recent trends in the lipid-based
electron microscopy. EC and VD wrote the manuscript; IA, JR ,and FM nanoencapsulation of antioxidants and their role in foods. J Sci
made a critical reading of the manuscript. Food Agric 86:2038–2045
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Compliance with Ethical Standards Jin X, Abel CA (2011) Comparison of major biocontrol strains of
non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins
Conflict of interest The authors declare that they have no conflict of and cyclopiazonic acid in maize. Food Addit Contam Part Chem
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