Jorge Rojas L Opez-Menchero, Minami Ogawa, Juan C. Mauricio, Juan Moreno, Jaime Moreno-García

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LWT - Food Science and Technology 144 (2021) 111250

Contents lists available at ScienceDirect

LWT
journal homepage: www.elsevier.com/locate/lwt

Effect of calcium alginate coating on the cell retention and fermentation of


a fungus-yeast immobilization system
Jorge Rojas López-Menchero a, Minami Ogawa a, b, Juan C. Mauricio a, Juan Moreno a,
Jaime Moreno-García a, *
a
Department of Agricultural Chemistry, Edaphology and Microbiology, Agrifood Campus of International Excellence CeiA3, University of Córdoba, Córdoba, Spain
b
Department of Food Science and Technology, University of California, Davis, Davis, CA, 95616, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Yeast cell leakage from the matrix is one of the reoccurring inconveniences of most yeast immobilization systems.
Yeast immobilization Yeast biocapsules are a cost-effective, spontaneous immobilization system whereby yeast cells are attached to the
Yeast biocapsules hyphae of a filamentous fungus, creating hollow spheres that allow recovery and reutilization. In an attempt to
Alginate coating
prevent yeast cell leakage from the fungal matrix, we coated the yeast biocapsules with a 0.2% (w/v) alginate
Fermentation
layer and evaluated yeast cell immobilization and the potential to ferment synthetic high sugar medium and
grape must. We concluded that alginate coating prevents cell leakage from yeast biocapsules but hinders alco­
holic fermentation. In grape must fermentations, alginate coating does not affect wine acidity but impacts
concentrations of some volatile compounds.

1. Introduction (Bleve et al., 2016).


Despite the advantages, industrial utilization of immobilized yeast
Whole-cell immobilization is defined as the process by which cells cells is still limited (Berbegal et al., 2017; Djordjevic et al., 2016, pp.
are confined to a certain region while preserving their catalytic activity 1–40). One of the main reasons is the high investment (economic and
(Karel et al., 1985). The immobilization of yeast cells offers numerous time) required to integrate these technologies into traditional practices
opportunities for industrial fermentation processes, such as alcoholic without secure outcomes (Moreno-García et al., 2018a). Nedović et al.
beverages or biofuel production. When compared to techniques using (2015) suggested that future research should be focused on yeast car­
free yeast cells, immobilization systems bear easier cell withdrawal and riers that can be stored for long periods and allow processes that are
reutilization, permit the enhancement and prolongation of certain simple, flexible, non-expensive, and can be readily scaled up. Moreover,
metabolite production, provide high resistance against shear forces and in the case of alcoholic beverages, other factors such as abundance,
inhibitor compounds, among other advantages (Norton et al., 1995; cost-effectiveness, and food-grade quality are also crucial for the con­
Najafpour et al., 2004; Bai et al., 2008; Takei et al., 2011; Nedović et al., sumers’ acceptance, safety issues, and profitability (Kourkoutas et al.,
2015; Yuvadetkun et al., 2018). 2004).
The most common yeast immobilization system is Ca-alginate gel In this context, an atypical method of immobilization, known as
beads (Moreno-García et al., 2018a). The popularity of this system in yeast biocapsules, emerges as a non-expensive, completely natural bio­
alcoholic fermentation is usually because of its ease of preparation under immobilization technology where yeast cells adhere to inactive fungal
gentle conditions (Clementz et al., 2015). For this method, yeasts are hyphae of a generally recognized as safe (GRAS) filamentous fungus
mixed in a Na + alginate solution and dripped into a solution with Ca2+ (Peinado et al., 2006). The yeast cells remain attached to the hyphae of
which causes the droplets to form spheres containing the cells (Smidsrod the fungus, forming hollow spheres with a porous wall that facilitates
and Skjak-Braek, 1990). Ca-alginate beads have been successfully tested mass transfer reactions. The use of using a food-grade, culturable, fila­
in cell-recycle batch process and optimization of primary must fer­ mentous fungus as support allows the system to be completely natural
mentations (Suzzi et al., 1996), sluggish and stuck fermentations (Silva and sustainable while improving ethanol yields and the aromatic profile
et al., 2002), or simultaneous alcoholic-malolactic wine fermentations of alcoholic beverages (García-Martinez et al., 2011, 2015).

* Corresponding author.
E-mail address: [email protected] (J. Moreno-García).

https://doi.org/10.1016/j.lwt.2021.111250
Received 9 December 2020; Received in revised form 4 March 2021; Accepted 5 March 2021
Available online 9 March 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
J.R. López-Menchero et al. LWT 144 (2021) 111250

Fig. 1. Yeast immobilization formats utilized in this study: alginate coated yeast biocapsules (AYB), yeast biocapsules (YB), alginate beads (AB), and free yeast
cells (FY).

Yeast biocapsules have been successfully tested for the production of shaken at 175 rpm, 28 ◦ C, for 6 d (Moreno-García et al., 2018c; Ogawa
bioethanol and alcoholic beverages such as white wine, sparkling wine, et al., 2020). In order to avoid fungal metabolism interference and to
natural sweet wine, and long-aged sparkling wines (Peinado et al., 2006; keep the hypha as a mere support, the filamentous fungus was inacti­
García-Martínez et al., 2012, 2015; Puig-Pujol et al., 2013; Lopez de vated by submerging YB into a high-sugar medium (YP + 25% dextrose)
Lerma et al., 2018; Martínez-García et al., 2020), but like other immo­ for 12 days (García-Martinez et al., 2011). After this process, half of the
bilization techniques, yeast biocapsules have an inherent flaw where YB were used to make AYB and the other half were kept without alginate
yeast cells release from the support. This problem becomes more coating.
obvious when yeast biocapsules are immersed in a sugar-rich medium AYB were made by coating the fresh YB with a layer made of 0.2%
when cells release from the fungal hyphae and reproduce unattached. (w/v) of sodium alginate according to specifications described in Hi­
In an attempt to prevent yeast cell leakage from the matrix, we dalgo (2011, pp. 1327–1329). The same protocol was used to prepare
coated the yeast biocapsules with a 0.2% (w/v) alginate layer and the AB using 2% (w/v) of sodium alginate, and 2% (w/v) of CaCl2. After
evaluated yeast cell leakage and the fermentation potential using syn­ preparing the beads, the AB were also coated with an alginate layer
thetic high sugar medium (for bioethanol production) and grape must made with 0.2% (w/v) of sodium alginate and 2% (w/v) CaCl2.
(for winemaking). These parameters were compared in biocapsules
without the alginate coat, yeast cells encapsulated in alginate beads, and 2.3. Fermentative conditions
yeast cells in suspended format. With this approach, we hypothesized
that the combination of alginate and yeast biocapsules will result in an Two fermentations were carried out: fermentation of a high-sugar
immobilization system that gathers both the natural composition and synthetic medium (SM) and a grape must (GM). The SM was
high substrate-product diffusion of yeast biocapsules, with the high composed of 1% yeast extract, 2% peptone, and 25% dextrose, and
immobilization yield provided by the Ca-alginate. fermentations were conducted for 10 day at 28 ◦ C without agitation in
100 mL Erlenmeyer flasks containing 50 mL of SM. The GM consisted of
2. Materials and methods juice with a sugar content of 220 g/L obtained from grapes of the Pedro
Ximénez variety grown in the Montilla-Moriles winemaking region
2.1. Microorganisms and growth media (Córdoba, south Spain). The must has a pH value of 4.29 ± 0.01, a
volatile acidity of 0.13 ± 0.02 (expressed as g acetic acid/L), and a
Saccharomyces cerevisiae G1 strain (ATCC: MYA-2451), a biofilm- titratable acidity of 2.02 ± 0.07 (expressed as g tartaric acid/L). The GM
forming yeast used in the biological aging of Sherry-type wines, and fermentations were carried out in 500 mL Erlenmeyer flasks containing
the filamentous fungus Penicillium chrysogenum H3 were used in this 250 mL of must at 21 ◦ C for ten days. All fermentations (YB, AYB, AB,
study. These two fungi strains have been previously proven to be good and FY) were run parallelly and monitored via weight loss of flask due to
candidates for co-adhesion (Moreno-García et al., 2018b; Peinado et al., the CO2 released during fermentation (Bezenger et al., 1985). All media
2006). In the case of yeast cell encapsulation in alginate, yeast cells were were inoculated with a concentration of 1 × 106 yeast cells/mL or the
streaked out on YPD agar (1% yeast extract, 2% peptone, 2% dextrose, correspondent wet weight in case of immobilization systems. Yeast cells
2% agar) and then cultured in YPD overnight at 175 rpm, 28 ◦ C (Hi­ in immobilization formats were subjected to a cell-carrier detachment
dalgo, 2011, pp. 1327–1329). In the case of yeast biocapsules, YPG (1% step previous to cell counting. In the case of YB and the AYB, cells were
yeast extract, 2% peptone, 3% glycerol) was used as a pre-culture me­ quantified by submerging five random immobilization spheres per BFM
dium and cultivated for 3 days at 175 rpm, 28 ◦ C (Moreno-García et al., flask in a solution of 0.1 M NaCl, disrupting them with a mortar and
2018b). The filamentous fungus was cultured on a sporulation medium pestle for 2 min, and vortexing for 20 min (Moreno-García et al., 2018b).
(1.7% corn meal agar, 0.1% yeast extract, 0.2% glucose, 2% agar). With this method, a mixture of detached yeast cells and P. chrysogenum
hyphae segments was obtained and yeast cells were easily distinguished
from the fungal mycelia under the microscope (40 × objective). In the
2.2. Yeast immobilization procedures case of AB, the alginate gel was dissolved in sodium 1% (w/v) citrate
solution under agitation and yeast cells were released (Hidalgo, 2011,
Yeast cells were immobilized in three different formats: yeast bio­ pp. 1327–1329). Cells were counted using a Thoma chamber under the
capsules (YB), alginate coated yeast biocapsules (AYB), and alginate microscope at 40 × objective. Samples were tested for contamination
beads (AB). free yeast cells (FY) were used as the control (Fig. 1). YB after each fermentation.
were obtained in a biocapsule formation medium composed of yeast
nitrogen base medium without amino acids (Difco), 5 g/L gluconic acid,
and buffered to pH 7 with Na2HPO4 and KH2PO4. In sterile, autoclaved
250 mL Erlenmeyer flasks containing 150 mL BFM, 4 × 106 yeast cells/
mL and 3 × 104 P. chrysogenum spores/mL were co-inoculated and

2
J.R. López-Menchero et al. LWT 144 (2021) 111250

Fig. 2. Yeast immobilization-related parameters before and after fermentation of synthetic medium (SM) (a–c) or grape must (GM) (d–e): number of yeast cells
immobilized and not immobilized per medium volume, per immobilization sphere and per g wet weight (WW) of alginate coated yeast biocapsules (AYB) in green,
yeast biocapsules (YB) in orange, or alginate beads (AB) in yellow. Darker colour bars represent yeasts immobilized and lighter colour bars represent non-
immobilized or free yeasts. Values after fermentation are indicated with brackets. The error bars and the value after the ± sign indicate the standard deviation.
Different letters above the bars indicate homogeneous groups statistically significantly differing in the parameters among the strains (p < 0.05, F-test). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.4. Measurement of yeast immobilization yields and enological an internal standard solution (1 g/L of 4-methyl-2-pentanol in 14% (v/v)
parameters ethanol) to 10 mL of wine. The initial oven temperature was set to 45 ◦ C
for 15 min, ramped to 190 ◦ C at 4 ◦ C min− 1, and held for 35 min. The
Yeast population sizes (non-immobilized and immobilized yeasts) temperatures of the injector and detector were 270◦ and 300 ◦ C,
were determined by direct cell-carrier separation step when required respectively. Helium was used as the carrier gas and injections were
(previously described) and cell counting using a Thoma chamber under done in split mode (1:10). The flow-rate program was as follows: initial
the microscope at 40 × objective. setting 0.7 mL min− 1, hold for 16 min, ramp to 1.1 mL min− 1 at 0.2 mL
Analytical parameters commonly used for alcoholic beverages min− 1, and hold for 52 min. The chromatographic peaks were assigned
(ethanol, total and volatile acidity and pH) were quantified applying to a specific compound by its mass spectrum and by the addition of pure
methods recommended by the International Organization of Vine and compounds provided by Fluka, Merck, and Sigma-Aldrich, and quanti­
Wine (OIV, 2012) for the non-fermented and fermented media. Glucose fication was performed by means of a calibration table built with stan­
and fructose were measured using the D-Fructose/D-Glucose Assay Kit dard solutions, containing known concentrations of the target
(Megazyme International Ireland, Bray, Ireland). compounds analyzed in wine samples (Vararu et al., 2016). The quan­
tified compounds were identified and confirmed by GC–MS by an Agi­
lent 7890 A with MSD-5975-C (Wilmington, DE, USA) using the same
2.5. Quantification of major aroma compounds and polyols capillary column and programs for temperature and helium.

Volatile aroma compounds and polyols that contribute to the


organoleptic properties of the resulting wine from the GM fermentation 2.6. Statistical analysis
were quantified by direct injection of wine samples in a Gas Chro­
matograph (GC) Agilent 6890 (Palo Alto, California, USA) provided with The significant differences among different immobilization condi­
a fused silica capillary column (60 m, 0.25 mm diameter, 0.4 μm film) tions were confirmed by multiple sample comparisons by homogenous
coupled to a Flame Ionization Detector (FID). Chromatographic condi­ groups (HG) and Multiple Variable Analysis (MVA) sunray plots using
tions were set according to the Peinado et al. (2004) method. The con­ Statgraphics v. XVI.I (StatPoint Technologies Inc., Warrenton, Virginia,
centration values of the wine compounds were obtained by direct USA). Principal component analysis (PCA) was obtained by PAST, v.3.23
injection in the GC inlet of 1 μL of a mixture prepared by adding 1 mL of (Hammer et al., 2001).

3
J.R. López-Menchero et al. LWT 144 (2021) 111250

Fig. 3. Yeast cells immobilized in alginate coated yeast biocapsules (AYB) in green, yeast biocapsules (YB) in orange, alginate beads (AB) in yellow, and free yeast
cells (FY) in blue observed under the microscope at a 40 × objective. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)

Fig. 4. Fermentation kinetics. Evolution of CO2 pro­


duction rate by alginate coated yeast biocapsules
(AYB) in green lines and circles, yeast biocapsules
(YB) in lines and orange squares, alginate beads (AB)
in yellow lines and triangles, and free yeast cells (FY)
in blue lines and crosses when fermenting a synthetic
medium (SM) (a) and grape must (GM) (b). The error
bars and the value after the ± sign indicate the stan­
dard deviation. (For interpretation of the references to
colour in this figure legend, the reader is referred to
the Web version of this article.)

3. Results biocapsule when coated with alginate (Fig. 2a and d). Yeast cell accu­
mulation in the immobilization matrices during fermentation was re­
3.1. Immobilization yield ported in all three studied systems, where AB was most prominent
(Fig. 2b, c, e, and f). Fig. 3 shows the yeast cell morphology before and
Yeast immobilization-related parameters are shown in Fig. 2. A 4- after fermentation for all immobilized formats. A higher frequency of
fold and 6-fold immobilization yield (immobilized:total cells) spherical cells carrying vacuoles were reported after fermentation
improvement was attained in AYB versus YB after SM and GM fermen­ specially in AYB and AB.
tation, respectively, indicating significant retention of cells in the yeast

4
J.R. López-Menchero et al. LWT 144 (2021) 111250

representing [CO2] produced over time, our data will retrieve a sigmoid
curve, typical of fermentation Gompertz’s equation and
Lineweaver-Burk plot (Callone et al., 2008).
Residual sugar contents <1 g/L (data not shown) confirm that all
fermentations were completed in all formats of immobilization. Ethanol
concentrations of the fermented media were analyzed for all conditions
(Fig. 5). It appears that ethanol production in AB was the lowest among
all studied formats: 5.57 ± 0.16% (v/v) in SM and 9.26 ± 0.18% (v/v) in
GM). The highest values were obtained when using YB (8.15 ± 0.61% in
v/v) in SM and FY (10.54 ± 0.32% in v/v) in GM but closely followed by
YB (10.39 ± 0.35% in v/v) in the last case. The alginate layer reduces the
YB ethanol production by 1.24% (v/v) in SM and 0.92% (v/v) in GM but
in both cases, the ethanol concentration was higher than AB. This fact
reflects a negative effect on ethanol production when using alginate.

3.3. Acidity and major volatile compounds

Acidity parameters obtained in wine by each method of immobili­


zation are represented in Table 1 pH was lower in the fermentations
carried out by AYB and YB while titratable, volatile, and fixed acidities
were higher.
Major volatile compounds of the finished wines are shown in Table 2.
The 13 identified and quantified major volatiles are by-products of
fermentation. Among them, methanol, acetaldehyde, and diethyl suc­
cinate did not show significant differences between conditions. The
highest concentrations were detected in the polyols, which includes 2,3-
butanediol (levo and meso) and glycerol and lowest in ethyl lactate. AB
displayed 8 compounds with the same HG as free yeast. This contrasts
with the YB that had 5 compounds in the same HG with FY and AYB with
4 compounds in the same HG as the FY. Propanol, isobutanol and ethyl
lactate were all higher in YB compared to FY which are consistent with
Fig. 5. Ethanol produced at the end of the fermentation by alginate coated results from previous studies (Garcia-Martinez et al., 2015). Comparing
yeast biocapsules (AYB) in green, yeast biocapsules (YB) in orange, alginate AYB to YB and also AYB to AB, 5 compounds shared the same HG for
beads (AB) in yellow, and free yeast cells (FY) in blue when fermenting a both conditions: methanol, acetaldehyde, ethyl acetate, ethyl lactate,
synthetic medium (SM) (a) and grape must (GM) (b). The error bars and the and diethyl succinate in the case of YB; and methanol, isoamyl alcohol,
value after the ± sign indicate the standard deviation. Different letters above
acetaldehyde, diethyl succinate, and glycerol in the case of AB.
the bars indicate homogeneous groups statistically significantly differing in the
All exometabolites quantified were placed on a sunray plot (Fig. 6) to
parameters among the strains (p < 0.05, F-test). (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web visualize the major volatile footprints of each condition. Further, the
version of this article.) exometabolites that had two or more HG between conditions were
graphed on a PCA with PC1 accounting for 57.5% and PC2 accounting
for 28.5% of the variance (Fig. 7). 2-phenylethanol, 1-propanol, acetoin,
3.2. Fermentation parameters
isobutanol, ethyl acetate, and ethyl lactate have positive projections
over PC1 while glycerol, 2-3-butanediol (meso and levo) are negative
Fig. 4 shows the fermentation rates in SM and GM, following Bely
over PC1. All compounds except ethyl acetate and ethyl lactate had
et al. (1990) kinetic models. The higher fermentation rates in SM were
positive projections over PC2. In this PCA, the YB and AYB conditions
attained by AYB closely followed by YB on day two of fermentation with
are positioned to the right while FY and AB conditions are positioned on
a maximum weight loss of 5.38 ± 0.62 and 4.45 ± 0.12 g CO2/day,
the left while YB and FY are above the x axis and AYB and AB below it.
respectively (equivalent to a production rate of 5.62 ± 0.65 and 4.65 ±
0.13 g ethanol/day respectively). As it is shown in Fig. 4, YB, AYB, and
FY fermentations concluded at day four while AB at day seven. The 4. Discussion
highest rate in GM fermentation was reached by the FY at day three with
a weight loss of 11.32 ± 0.5 g CO2/day (11.8 ± 0.52 g ethanol/day). In 4.1. Alginate coating prevents cell leakage from yeast biocapsules but
this medium, fermentations with FY inoculum ended at day five, AB at hinders alcoholic fermentation
day seven while YB and AYB at day nine. Yeast cell immobilized in YB
and AYB fermented at high rates for longer than AB or FY. If Alginate is a natural anionic polysaccharide composed of alternating
repeated residues of d-mannuronate and l-guluronate with (1–4)

Table 1
Acidity parameters in wines made with alginate coated yeast biocapsules (AYB), yeast biocapsules (YB), alginate beads (AB), and free yeast cells (FY).
Acidity parameters AYB YB AB FY

Avg SD HG Avg SD HG Avg SD HG Avg SD HG

pH 3.77 0.02 a 3.80 0.10 A 4.08 0.02 b 4.01 0.02 b


Titratable acidity (g H2T/L) 3.32 0.04 c 3.10 0.26 C 1.90 0.13 a 2.46 0.06 b
Volatile acidity (g ACH/L) 0.61 0.09 c 0.56 0.11 C 0.36 0.04 b 0.40 0.02 b
Fixed acidity (g ACH/L) 2.72 0.07 c 2.54 0.22 C 1.39 0.26 a 2.06 0.07 b

Different letters indicate homogeneous groups statistically significantly differing in the parameters among the strains (p < 0.05, F-test).

5
J.R. López-Menchero et al. LWT 144 (2021) 111250

linkages. It is a commonly used material to encapsulate whole cells and

Different letters indicate homogeneous groups statistically significantly differing in the parameters among the strains (p < 0.05, F-test). CAS number, Odor Threshold (OT), Flavor Threshold (FT), and Odor/flavor
HG
enzymes because of its ease of preparation and no need to use extreme

b
a

a
a

a
a

a
c

c
conditions (Clementz et al., 2015). Other than encapsulation, alginate

560.09
has been efficiently proven for sealing or coating other immobilization

15.68

30.38
3.67
1.33
0.62
7.95

4.24

4.21

0.35
0.00

1.06

6.87
Major volatile compounds production in wines made with alginate coated yeast biocapsules (AYB), yeast biocapsules (YB), alginate beads (AB), and free yeast cells (FY) when fermenting grape must (GM).

description are provided for each compound if available. Shadowed cells indicate those concentrations that are above the compound odor threshold. Threshold values are taken from Moreno et al. (2016).
systems, such as glass pellets, alginate beads, and carboxymethyl cel­
SD

lulose (Ogbonna et al., 1989; Uemura et al., 2000; Yinzhe & Shaoying,
Average

2013). Here, we studied for the first time, the effect of calcium alginate
108.27

221.27

192.62

847.11

274.37

125.22
31.68
35.47

66.33

55.65

12.45

19.63
0.00
coating on the performance of immobilized yeast cells in a fungus-yeast
FY

immobilization system, the yeast biocapsules. As observed in Fig. 2,


alginate efficiently coats the surface of yeast biocapsules, which is
HG

ab

b
a
a
a
a

a
a

a
c
mainly composed of glycoproteins and carbohydrates, and prevents
yeast cells from leaking from the yeast biocapsule system during

104.96
15.09

60.73
24.12

33.88

15.82
9.52
3.51
1.92

5.30

2.06
0.00

2.45
sugar-rich medium and a grape must fermentation.
SD

Although detachment is minimized, alginate coating limits the pro­


duction of ethanol and other fermentation by-products like higher al­
Average

155.27

137.99

864.93

280.85
85.03
20.47
19.24

34.96

62.36

32.82

15.18

90.51
cohols, acetaldehyde and derivatives, and some esters and polyols
0.00
AB

(Figs. 5 and 6 and Table 2). Lebeau et al. (1998) reported limits on mass
transfer reactions when using solid alginate matrices that hinder me­
HG

b
b

b
a

a
c

tabolites diffusion through the gel. These limitations can decelerate the
consumption of nutrients and excretion of toxic compounds. In this
30.05

37.96

11.83

71.89

14.32
3.80
0.81
0.67

4.25

6.21

0.63

2.63

7.61

study, we observed that this limitation becomes more apparent in the AB


SD

(Figs. 5 and 6 and Table 2) thus, supporting the hypothesis that alginate
Average

polysaccharide limits mass transfer reaction. Low mass transfer leads to


169.36
191.52

190.06

201.62

651.44

274.78

109.30
81.81
54.29

80.32

92.29

35.80
9.98

an accumulation of dead cells in the core of the beads and results in a


YB

large number of cells without catalytic activity (Duarte, Gaudreau, et al.,


2013).
HG

bc
b

b
b
a

a
c

Another potential reason is that yeast cells perform oxidative meta­


bolism apart from fermentative thus promoting biomass growth and
19.14
16.57

18.47
16.52

59.10

62.30

39.23
8.10
2.05

7.84

2.83

2.06

6.79

storage rather than production of fermentative related metabolites. This


SD

hypothesis is supported by (i) the total sugar consumption which in­


Average

dicates that the lower concentration of fermentation products are due to


140.90
162.30

185.31

179.13

487.42

219.52
88.74
42.09

61.96

86.72

10.01

29.29

88.08
AYB

differences in the metabolism rather than dissimilarities in sugar con­


sumption (data not shown), (ii) the high number of yeast cells retained
in the alginate matrix in AB once the fermentation is over (Fig. 2) and
Alcohol, wine like, nail polish

Confers body and smoothness


Pineapple, varnish, balsamic

(iii) the high frequency of vacuoles in the AB immobilized cells that can
Odor/flavour description

be functioning as storage organelles. This is reasonable since the Crab­


Strawberry, raspberry,
Chemical, medicinal

Alcohol, nail polish

Over-ripe, lavender

tree effect could be induced less during early stage alginate beads when
Ripe fruit, alcohol

and a sweet taste


Buttery, creamy

Buttery, creamy
Over-ripe apple

the ratio yeast biomass:alginate mass is lower and sugars reach cells in a
Buttery, cream

slower motion (Wunderlich & Back, 2009, pp. 3–16). S. cerevisiae under
fermentative conditions usually converts 95% of the sugar into ethanol
buttery

and CO2, 1% in cellular material, and 4% in other products like higher


N.f

alcohols, esters and aldehydes (Chidi et al., 2018); while under oxidative
metabolism the yeast mostly converts the sugars into cell material. High
4400000
FT (μg/

microbial loads ease clarification processes and circumvent collapse of


4800
170

filters that can be beneficial under industrial settings, but excessive


L)

loads destabilize the alginate beads and eventually provoke their


(mg/L)

rupture, releasing yeast cells and alginate particles to the medium


668
830

100

100

668

668

(Duarte, Gaudreau, et al., 2013; Clementz et al., 2015; Karagoz et al.,


7.5
OT

40
30

10

10
30

2019).
123-51-3

141-78-6

123-25-1

5341-95-
67-56-1
71-23-8
78-83-1

60-12-8

75-07-0

97-64-3

56-81-5
53584-

24347-

4.2. Alginate coating in yeast biocapsules does not affect acidity but
56-8

58-8
CAS

concentration of volatile compounds in wines


7
2,3-butanediol (meso)
2-phenylethanol (mg/
Isoamyl alcohol (mg/

Acetaldehyde (mg/L)

2,3-butanediol (levo)

Glycerol (dg/L)*0.01
Ethyl acetate (mg/L)

The pH in all of the samples had a diminution over the non-


Ethyl lactate (mg/L)
1-Propanol (mg/L)
Isobutanol (mg/L)
Methanol (mg/L)

Diethyl succinate

fermented medium as a result of the yeast metabolic activity


Acetoin (mg/L)

(Table 1). During the fermentation, the microorganisms present in the


grape must produce acids, mostly succinic, lactic and acetic (volatile
(mg/L)

(mg/L)

(mg/L)

acidity), and also other acids in smaller amounts (Mato et al., 2005).
L)

L)

Therefore, the lower pH values exhibited by YB and AYB are due to a


Major volatile compounds

higher acid production because of changes in the metabolism, probably


as a consequence of the method of immobilization.
Acetaldehyde and

Titratable acidity increased in all cases except for AB (Table 1). The
use of calcium-alginate beads for winemaking causes accumulation of
derivative

Ca2+ in the wine (de Lerma et al., 2018; Puig-Pujol et al., 2013; López).
Alcohols

Polyols
Table 2

Esters

This accumulation interferes with some compounds present in the wine.


One of them is tartaric acid, causing its precipitation as calcium tartrate

6
J.R. López-Menchero et al. LWT 144 (2021) 111250

Fig. 6. Multiple Variable Analysis (MVA) sunray plots


for the concentration of major volatile compounds
produced by alginate coated yeast biocapsules (AYB)
in green lines and circles, yeast biocapsules (YB) in
orange lines and squares, alginate beads (AB) in yel­
low lines and triangles, and free yeast cells (FY) in
blue lines and crosses when fermenting grape must.
The end of the ray is the mean value plus three stan­
dard deviations and the center the mean minus three
standard deviations. (For interpretation of the refer­
ences to colour in this figure legend, the reader is
referred to the Web version of this article.)

Fig. 7. Multiple Variable Analysis (MVA) Principal Component Analysis (PCA) for the concentration of major volatile compounds produced by alginate coated yeast
biocapsules (AYB) in green, yeast biocapsules (YB) in orange, alginate beads (AB) in yellow, and free yeast cells (FY) in blue when fermenting grape must. Principal
Components Analysis Biplot was carried out with ten compounds selected by their discrimination power among the immobilization systems studied. (For inter­
pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

which may explain the lower titratable acidity observed. This event may The first two volatiles, isoamyl alcohol and 2-phenylethanol, are
lead to wine browning and deposits of calcium tartrate in wine higher alcohols which are produced from sugars and from amino acids
(Darias-Martin et al., 2000). This effect did not appear in the AYB, during the alcoholic fermentation process (Guymon & Heitz, 1952;
probably because of the low amount of alginate (0.2% (w/v) in AYB Zoecklein et al., 1955, pp. 101–104). Isoamyl alcohol can give off odors
versus 2% (w/v) in AB) used to coat the biocapsules. of alcohol and nail polish while 2-phenylethanol is reminiscent of rose
Regarding the volatile acidity, all obtained values were below 1.2 g and honey. Another higher alcohol, isobutanol, was detected over its
of acetic acid/L which is the maximum acceptable concentration for threshold limit only in AYB and YB conditions and gives off odors of
wine established by the International Organization of Vine and Wine or wine-like alcohol or nail polish. The higher concentration of isobutanol
OIV (OIV, 2011). A high volatile acidity can produce a vinegar-like in YB is consistent with previous findings (Peinado et al., 2004; Gar­
flavor to the wine (Swiegers et al., 2005) an undesirable effect for the cia-Martinez et al., 2015). In normal wine composition, total higher
wine product. Even far below 1.2 g of acetic acid/L, highest volatile alcohol does not have strong sensory effects due to its high threshold and
acidity was reported in AB and AYB fermentations. Erasmus et al. (2004) are normally found in wines at concentration from 100 to 500 mg/L
reported that under stress conditions produced by high sugar, the yeasts (Boulton et al., 1999). Since the total higher alcohol content in each
abnormally produce higher acetic acid. This, in addition to the higher condition is within this range, these volatiles probably do not have a
stress of the yeast contained in YB and AYB because of the necessity of a high impact on the overall aroma bouquet.
phase for killing the fungus in a high-sugar medium, could produce The next two volatiles, acetaldehyde, and acetoin are important
metabolic changes in the immobilized yeast cells, producing more vol­ sensory carbonyl compounds in wine. Acetaldehyde gives off enjoyable
atile acidity in the obtained wine. fruity aromas at low concentrations and overripe apples at high con­
The major volatiles that arise from Saccharomyces fermentation centrations (over 125 mg/L) (The Australian Wine Research Institute,
contribute to the wine aroma and flavor, an important aspect of finished Moreno-García et al., 2015). Acetaldehyde production is specific to
wines that can be positive, negative, or neutral depending on the com­ yeast species and is thought to be a leakage product of alcoholic
pound and its concentration. If a compound concentration surpasses its fermentation (Liu & Pilone, 2000). In this study, the high levels of
threshold detection limit, the compound can have an impact on wine acetaldehyde seen in all conditions most likely due to the use of flor
aroma. In this study, the major volatile compounds that exceeded its yeast strain G1 (García-Martínez et al., 2015). For acetoin, slightly lower
threshold limit in all conditions were isoamyl alcohol, 2-phenylethanol, concentrations were seen in FY and AB which contributes to aromas that
acetaldehyde, acetoin, and ethyl acetate (Table 2). are buttery or creamy, while concentrations of 2,3-butanediol, also

7
J.R. López-Menchero et al. LWT 144 (2021) 111250

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