Jorge Rojas L Opez-Menchero, Minami Ogawa, Juan C. Mauricio, Juan Moreno, Jaime Moreno-García
Jorge Rojas L Opez-Menchero, Minami Ogawa, Juan C. Mauricio, Juan Moreno, Jaime Moreno-García
Jorge Rojas L Opez-Menchero, Minami Ogawa, Juan C. Mauricio, Juan Moreno, Jaime Moreno-García
LWT
journal homepage: www.elsevier.com/locate/lwt
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.
* 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
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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.
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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.)
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.
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
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
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
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
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
(Figs. 5 and 6 and Table 2) thus, supporting the hypothesis that alginate
Average
190.06
201.62
651.44
274.78
109.30
81.81
54.29
80.32
92.29
35.80
9.98
bc
b
b
b
a
a
c
18.47
16.52
59.10
62.30
39.23
8.10
2.05
7.84
2.83
2.06
6.79
185.31
179.13
487.42
219.52
88.74
42.09
61.96
86.72
10.01
29.29
88.08
AYB
(iii) the high frequency of vacuoles in the AB immobilized cells that can
Odor/flavour description
Over-ripe, lavender
tree effect could be induced less during early stage alginate beads when
Ripe fruit, alcohol
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
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/
100
100
668
668
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
Acetaldehyde (mg/L)
2,3-butanediol (levo)
Glycerol (dg/L)*0.01
Ethyl acetate (mg/L)
Diethyl succinate
(mg/L)
(mg/L)
acidity), and also other acids in smaller amounts (Mato et al., 2005).
L)
L)
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
6
J.R. López-Menchero et al. LWT 144 (2021) 111250
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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