See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/328511244

Sequential inoculum of Hanseniaspora guilliermondii and Saccharomyces

cerevisiae for winemaking Campanino on an industrial scale

Article  in  World Journal of Microbiology and Biotechnology · November 2018

DOI: 10.1007/s11274-018-2540-6

CITATIONS READS

0 63

10 authors, including:

Silvia Jane Lombardi Massimo Iorizzo

Università degli Studi del Molise Università degli Studi del Molise
20 PUBLICATIONS   69 CITATIONS    72 PUBLICATIONS   595 CITATIONS   

SEE PROFILE SEE PROFILE

Elena Sorrentino
Università degli Studi del Molise
133 PUBLICATIONS   1,583 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Approcci biotecnologici per la valorizzazione del tartufo del Molise - Biotechnological approaches for the valorization of the truffle of Molise (Italy) View project

Valorizzazione di prodotti da acquacoltura in Campania View project

All content following this page was uploaded by Silvia Jane Lombardi on 17 November 2018.

The user has requested enhancement of the downloaded file.

World Journal of Microbiology and Biotechnology (2018) 34:161
https://doi.org/10.1007/s11274-018-2540-6

ORIGINAL PAPER

Sequential inoculum of Hanseniaspora guilliermondii

and Saccharomyces cerevisiae for winemaking Campanino
on an industrial scale
Silvia Jane Lombardi1 · Gianfranco Pannella1 · Massimo Iorizzo1   · Maria Victoria Moreno‑Arribas2 ·
Patrizio Tremonte1 · Mariantonietta Succi1 · Elena Sorrentino1 · Vincenzo Macciola1 · Massimo Di Renzo1,3 ·
Raffaele Coppola1

Received: 28 June 2018 / Accepted: 1 October 2018

© Springer Nature B.V. 2018

Abstract
In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomy-
ces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three
independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one
was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or
no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines
were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with
S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential
inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally,
sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest
that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice
that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale
H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.

Extended author information available on the last page of the article

13
Vol.:(0123456789)
 161   Page 2 of 10
Graphical abstract 
Keywords  Campanino white wine · Sequential inoculation · Non-Saccharomyces · H. guilliermondii · Industrial-scale
winemaking
Introduction
Grape must and wine are clearly described as hard environ-
ments (Testa et al. 2014; Tremonte et al. 2017a). However,
they arbour a complex microbiota including Saccharomyces
and non-Saccharomyces yeasts, as well as lactic acid bac-
teria, which is considered by winemakers and oenologists
as a decisive factor influencing wine aroma and consumers’
preferences (Iorizzo et al. 2016; Belda et al. 2017; Brizuela
et al. 2017; Succi et al. 2017a). S. cerevisiae strains are
mainly responsible for the alcoholic fermentation of grape
musts, yet there are also other non-Saccharomyces yeast spe-
cies present in grape juice that can influence the first step of
alcoholic fermentation, the composition and organoleptic
quality of the final wines (Ciani et al. 2010; Medina et al.
2013; Garcìa et al. 2016). The spontaneous fermentation
of the grape must can involve many wild yeasts (Candida,
Pichia, Metschnikowia, Brettanomyces, Saccharomycodes,
Zygosaccharomyces and Hansenula genera), with the initial
prevalence of apiculate yeasts, belonging to the Kloeckera
and Hanseniaspora genera, present on the grape or on the
equipment used and the winery environment (Fleet 2008;
Jolly et al. 2014; Lleixà et al. 2016). Industrial wine fer-
mentations are currently conducted by the use of commer-
cial starters cultures of yeasts. However, a never-ending
debate between researchers and oenologists is that the wine
13
World Journal of Microbiology and Biotechnology (2018) 34:161
produced with pure yeast monocultures lacks the complexity
of flavour, stylistic distinction and vintage variability cre-
ated by indigenous yeasts (Andorrà et al. 2010; Moschetti
et al. 2016). Therefore, apiculate yeasts have become a mat-
ter of great interest, especially for their growth in musts in
competition with S. cerevisiae and for their impact on the
sensory quality of wine, in view of their high ester produc-
tion. Non-Saccharomyces yeasts improve the wine flavor,
but are not able to complete fermentation due to their low
alcohol tolerance; in fact, for this reason, several authors
have studied fermentation with mixtures of yeasts, simulta-
neously used (Comitini et al. 2011; Lombardi et al. 2015;
Tristezza et al. 2016).
Nowadays it is widely accepted that the selection of non-
Saccharomyces strains through appropriate screening can
positively influence the winemaking process (Padilla et al.
2016; Wang et al. 2016). In particular, non-Saccharomyces
yeasts have several desired oenological characteristics that
are absent in S. cerevisiae, such as the production of high
levels of flavor compounds (Ferreira et al. 2001; Mendoza
and Farìas 2010; Liu et al. 2016). Different studies have
shown that in spontaneous fermentations, Saccharomyces
and non-Saccharomyces yeasts do not passively coexist,
on the contrary, they appear to interact. The benefit of the
metabolic properties of the non-Saccharomyces yeasts could
be important in winemaking, thus the interactive behavior
World Journal of Microbiology and Biotechnology (2018) 34:161 Page 3 of 10  161
between the different species needs to be considered and
studied in more detail (Zohre and Erten 2002; Belda et al.
2016). In this context, the inclusion of non-Saccharomyces
in wine, as part of mixed starters cultures together with
S. cerevisiae yeast has been suggested as a way of taking
advantage of the spontaneous fermentations, enhancing wine
aroma and complexity (Liu et al. 2016). However, this prac-
tice is linked to new challenges for researchers and oenolo-
gists. For example, the selection of suitable non-Saccharo-
myces strains, the appropriate modality of inoculation and
the yeast interactions, may modify wine composition playing
a fundamental role in the wine’s final quality (Ciani et al.
2016; Belda et al. 2017). Moreover, in the search for poten-
tial wine yeasts for use in wine production, autochthonous
or locally selected wine strains are preferable because these
yeasts are well adapted to the micro-conditions of the wine
generating a unique regional characteristic. Consequently
this research aims to evaluate the effect of the autochtho-
nous Hanseniaspora guilliermondii LS77 strain, used in
sequential inoculum with S. cerevisiae AM37 commercial
strain and confirmed the contribution of the strain LS77 to
sensory profile in Campanino wine, a typical white wine
from Molise region.
Materials and methods
Yeasts and growth conditions
In this study, two strains belonging respectively to S. cer-
evisiae and H. guilliermondii were used. In detail, S. cer-
evisiae AM37 is a commercial strain (Enobiotech, Novara
- Italy) while H. guilliermondii LS77, previously isolated
from Molise region grape musts and selected for its good
oenological properties (Iorizzo 1997), was from the Food
Microbiology culture collection of the DiAAA (Department
of Agricultural, Environmental and Food Science, Univer-
sity of Molise). S. cerevisiae AM37 strain was rehydrated
before use as required by the producer. While, a pre-culture
of the H. guilliermondii LS77 strain, after aerobic growth
at 20 °C for 48 h in YPD (2% w/v yeast extract, 2% w/v
peptone and 4% w/v dextrose), was used. The cells were
centrifuged at 10,000 rpm for 15 min at 4 °C, washed twice
with sterile water before to use.
Industrial scale winemaking design
For industrial scale winemaking autochthonous white
grapes of Molise Vitis vinifera cv Campanino were used.
The grapes were harvested during the 2015 vintage and
immediately transported to an industrial winery (VI.NI.CA
s.r.l.) located in the Molise region (Italy). The grape must,
used for the experiments, showed the following chemical
composition: pH 2.89; sugar content 210 g L−1, total acid-
ity 10 g L−1. Previous tests to determine the best timing
and yeast concentration for inoculation experiments were
made (data not shown). Then, industrial wine experiments
were performed in three batches: batch A (sequential inoc-
ulum) was inoculated with H. guilliermondii LS77 at a
concentration of 6.0 log CFU mL−1 and after 3 days of
alcoholic fermentation an inoculum of 6.0 log CFU m ­ L−1
of S. cerevisiae AM37 strain was conducted (Ferraro
et al. 2000); batch B was inoculated only with S. cerevi-
siae AM37; and batch C was obtained without any yeast
inoculum (spontaneous fermentation).
For each batch three replicates were carried out in three
stainless steel tanks (AISI 304 quality) with a working
volume of 9 hL. The industrial fermentation took place
according to the usual white wine winemaking. Spe-
cifically, before fermentation, potassium metabisulphite
(Scharlau, Chemie S. A.) was added in a concentration of
50 mg L−1. All batches were equipped with a jacket for
temperature control (20 ± 2 °C). H. guilliermondii LS77
and S. cerevisiae AM37 yeasts were used, with 5% of pied
de cuve, using an overnight cell culture added to the Cam-
panino grape must.
In all the batches, the fermentation process was moni-
tored, assessing the reducing sugars and ethanol content,
considering the variation during fermentation time. During
and after alcoholic fermentation, samples of each batch were
taken to analyse. Each sample was analysed in triplicate.
Microbiological analysis
The wine samples from each batch during the alcoholic fer-
mentation were collected in sterile bottles (at 0, 3, 6 and
9 days) and subjected to yeast enumeration and identifica-
tion. After they had been diluted, viable cell counts were
evaluated by the plate-counting technique using WL agar
(Oxoid, Unipath Ltd, Hampshire, England), which allows
to enumerate both Saccharomyces and non-Saccharomyces
yeasts. The colony color and colony topography parameters
were adopted to differentiate the Saccharomyces from non-
Saccharomyces and also to distinguish Hanseniaspora from
other non-Saccharomyces yeasts (Cavazza et al. 1992; Pall-
mann et al. 2001).
The plates were incubated at 28 °C for 3–4 days and
then the various colony types on WL Nutrient Agar were
enumerated. To verify the predominance or the survival of
the starter culture until the end of the observation period,
three to five colonies were picked at random from the high-
est diluted plates of WL. Overall, 100 colonies were col-
lected from WL agar plates. The yeasts were characterized
by RAPD-PCR as described by Cocolin et al. (2004) and by
Reale et al. (2013).
13
 161   Page 4 of 10 World Journal of Microbiology and Biotechnology (2018) 34:161

Composition and volatile compounds the analysis
The pH, total acidity (g L−1 as tartaric acid), tartaric acid
(g L−1), acetic acid (g L−1), total ash (g L−1), alkalinity ashes
(meq L−1), l-malic acid (g L−1), l-lactic acid (g L−1), d-glu-
conic acid (g L−1), dry extract (g L−1), catechins (mg L−1),
reducing sugar (g L−1), alcohol (% v/v) and glycerol (g L−1)
were analyzed according to the corresponding EC methods
(European Community 1990). Total phenols (mg L−1) were
determined spectrophotometrically by the Folin–Ciocalteu
method, using gallic acid as the standard (Singleton et al.
1999; Succi et al. 2017b). The volatile compounds (mg L−1)
were determined by gas chromatography (GC) (Thermo-
quest Mod. 8000—Rodano, Milan, Italy) and flame ioniza-
tion detection according to the method Iorizzo et al. (2014)
and De Leonardis et al. (2018).
To determine higher alcohol content, the wine was pre-
liminarily treated with calcium hydroxide (12%, w/v) in ratio
1:1 (v/v), then centrifuged at 4000 rpm for 5 min and neu-
tralized at pH 7.00 with HCl 0.5 N. Butan-2ol (0.1 mg mL−1
in water) was added as internal standard. GC analysis con-
ditions were the following: flame ionization detector at
260 °C; injections in split mode (split ratio 50:1); injection
port at 250 °C; oven program from 35 °C (10 min) to 240 °C
at a rate of 8 °C min−1; carrier gas helium with a flow rate
of 50 kPa.
Sensory analysis
Sensory analysis of wines was carried out by a panel group
composed of 15 professional tasters from the National
Organization of Wine Tasters (ONAV, Italy). The wines
obtained were assessed for the following attributes: aromatic
intensity, persistence, softness, herbaceous, fruit and floral,
acetic, reduced, oxidized, astringent, overall evaluation,
in accordance with the procedure specified in the official
ONAV testing sheet. The intensity of each attribute was
rated on a scale of zero to five. All the samples were tested
in one session and the 11 wine attributes were evaluated by
each taster according to ISO 4121:2003. The final punctua-
tion/score was obtained as the mean of the three evaluations
with their respective standard deviation.
Statistical analysis
All data are expressed as means ± SD of three measurements.
Microbial count levels, chemical and sensorial parameters
were analyzed by a General Linear Model based on ANOVA
(IBM SPSS Statistics 21). The post-hoc Bonferroni test was
used for pairwise comparison (Tremonte et al. 2017b). Sta-
tistical significance was attributed to values of P ≤ 0.05.
Results
Yeast population kinetics during industrial‑scale
fermentations
Microbiological results evidenced that the grape must was
initially characterized by the presence of both Saccharo-
myces and non-Saccharomyces yeasts with values of about
2.0 ± 0.3  log  CFU  mL −1 and 2.1 ± 0.2  log  CFU  mL −1,
respectively. Significant differences in yeast levels were
found depending on the diverse batches (A, B and C) and
the different sampling time. As expected, the highest count
levels of S. cerevisiae were detected in batch B and in batch
A, inoculated with the strain S. cerevisiae AM37, at time 0
and at 3rd day, respectively (Table 1). However, for batches
A and B the highest values in Saccharomyces levels, of
about 9.0 log CFU mL−1, were observed on the 6th day of
fermentation. On the other hand, the samples from the three
tanks belonging to batch C showed the lowest values in

Table 1  Evolution of Yeast population (Log CFU mL−1)* Time Batch A Batch B Batch C

Saccharomyces and non- (days)**
Saccharomyces yeasts during
winemaking fermentation in: Saccharomyces cerevisiae 0 1.9 ± 0.2Aa 6.9 ± 0.8Ba 2.0 ± 0.3Aa
batch A: H. guilliermondii
3 6.6 ± 0.8Ab 8.0 ± 0.8Bb 2.3 ± 0.3Ca
LS77 + S. cerevisiae AM37
(sequential inoculum); batch 6 8.9 ± 0.8Ac 9.0 ± 0.9Ac 6.1 ± 0.3Bb
B: S. cerevisiae AM37 9 6.4 ± 0.3Ab 6.5 ± 0.5Aa 6.0 ± 0.5Bb
(monoculture); batch C: Non-Saccharomyces–Hanseniaspora spp.*** 0 7.1 ± 0.8Aa 2.0 ± 0.2Ba 2.1 ± 0.2Ba
spontaneous fermentation
3 7.9 ± 0.8Ab 2.0 ± 0.2Ba 6.1 ± 0.3Cb
6 3.0 ± 0.2Ac 2.0 ± 0.2Ba 4.0 ± 0.2Cc
9 2.0 ± 0.2Ad 2.0 ± 0.2Aa 1.8 ± 0.2Aa

*Results were expressed as mean of three experiments ± SD

**A–C: within a row, different letters indicate significant differences (P ≤ 0.05); a–d: within a column, dif-
ferent letters indicate significant differences (P ≤ 0.05)
***All non-Saccharomyces colonies have been considered as Hanseniaspora spp. due to colony morphol-
ogy and color (Cavazza et al. 1992; Pallmann et al. 2001)

13
World Journal of Microbiology and Biotechnology (2018) 34:161 Page 5 of 10  161

Saccharomyces levels. In fact, in this batch the highest values

Table 2  Reducing sugars and alcohol content during the fermentation in batch A: H. guilliermondii LS77 + S. cerevisiae AM37 (sequential inoculum); batch B: S. cerevisiae AM37 (monocul-

4.61 ± 0.21b

11.87 ± 0.71b
0.15 ± 0.13a
0.15 ± 0.14a

12.50 ± 0.73a
12.40 ± 0.73a
in Saccharomyces level was about 6.1 ± 0.3 log CFU mL−1.
As for non-Saccharomyces the highest value was revealed in
batch A where the sequential inoculum was carried out. For

9
this batch, the initial value of about 7.1 ± 0.8 log CFU mL−1
due to the H. guilliermondii LS77 inoculum, was increased

4.60 ± 0.18b

12.20 ± 0.71b
0.20 ± 0.20a
0.23 ± 0.19a

12.45 ± 0.60a

11.87 ± 0.67c
up to 7.9 ± 0.8 log CFU mL−1 on the 3rd day of fermentation
with a significant decrease (until 2.0 ± 0.2 log CFU mL−1)
on the 9th day. In batch B non-Saccharomyces load showed

8
constant levels of about 2.0 ± 0.2 log CFU mL−1. Whereas,
the non-Saccharomyces levels in batch C (also in this case

9.00 ± 0.12b
12.00 ± 0.08b

12.07 ± 0.70b
11.59 ± 0.68b
5.00 ± 0.15a

12.39 ± 0.58a
belonging to Hanseniaspora spp.) showed significant dif-
ferences depending on the diverse sampling time. Specifi-
cally, a significant increase was observed on the 3rd day of

7
fermentation and a decrease (P ≤ 0.05) was detected on the

22.00 ± 0.15b

11.65 ± 0.61b
12.00 ± 0.10a

27.00 ± 0.16c

12.10 ± 0.65a

11.12 ± 0.64c
6th and 9th day of fermentation.
In all the batches the non-Saccharomyces colonies were
considered as Hanseniaspora spp. due to their morphol-
ogy and color. The microbial typization by RAPD-PCR

6
performed on yeasts isolated from all batches sustained

41.00 ± 0.12b

10.48 ± 0.67b
30.00 ± 0.13a

50.00 ± 0.10c

11.16 ± 0.65a

9.92 ± 0.62c
the effectiveness of the inoculation and the ability of the
strains to dominate or to survive during the fermentation
period. In fact, all the presumptive S. cerevisiae isolated
from batches A and B showed a coefficient of similarity

5
higher than 92% with S. cerevisiae AM37, used as inoculum

60.00 ± 0.19b

9.30 ± 0.62b
50.00 ± 0.10a

70.00 ± 0.09c

9.92 ± 0.66a

6.63 ± 0.61c
(data not shown). Similar results were obtained for H. guil-
liermondii, actually, all the isolates from batch A, considered
as H. guilliermondii, showed coefficients of similarity higher
4

than 90% with the strains H. guilliermondii LS77 used as

inoculum (data not shown).
172.00 ± 0.16b

2.36 ± 0.60b
160.00 ± 0.21a
157.00 ± 0.10a

3.10 ± 0.67a
3.28 ± 0.66a
Oenological characters of the wines

*a–c: within a row, different letters indicate significant differences (P ≤ 0.05)

3

Date of residual sugar and alcohol content are reported in

Table 2. Tanks related to batches A (sequential inoculum)
187.00 ± 0.20b

1.42 ± 0.51b
190.00 ± 0.21a

192.00 ± 0.21a

1.24 ± 0.50a

1.12 ± 0.58c
and B (monoculture) finished the fermentation leaving in Data are means ± standard deviations of three separate replicates
the must < 0.5 g L−1 of residual sugar already on the 8th day
of fermentation. In batch C (spontaneous fermentation) an
arrest of alcoholic fermentation was observed. In fact, the
2

values of about 4.6 g L−1 of reducing sugar was consistently

200.00 ± 0.15b

0.62 ± 0.66b
203.00 ± 0.18a

204.00 ± 0.19a

0.43 ± 0.62a

0.37 ± 0.55c

detected from the 8th day the of fermentation (Table 2) and

remained unchanged also on the days following the 9th day
ture); batch C: spontaneous fermentation

(data not shown). As expected, data related to alcohol forma-

tion highlighted a clear connection with sugar consumption.
1

The alcohol content, determined on the 9th day, in batch

210.00 ± 0.15a*

A wine reached 12.50% v/v (± 0.73), in batch B 12.40%

210.00 ± 0.18a
210.00 ± 0.17a

0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
Time (days)

v/v (± 0.73) while in batch C it was 11.87% v/v (± 0.71).

Reducing sugars (g L )
−1

In addition, several differences among the batches in both

residual sugar and alcohol content were detected during the
0

Alcohol (vol%)

fermentation period. Batch C, during all the fermentation

period showed the lowest values in alcohol level and the
 Batch A

 Batch A
 Batch B
 Batch C

 Batch B
 Batch C

highest in sugar residual, indicating an alcoholic fermen-

tation behaviour less performant than that detected in the

13
 161   Page 6 of 10 World Journal of Microbiology and Biotechnology (2018) 34:161

other two batches. Moreover, significant differences were day (corresponding to the inoculum of S. cerevisiae AM37
detected between batches A and B. To be more specific, in batch A) the samples from batch A highlighted value in
during the first 2 days, in the samples from batch A residual residual sugar lower than the samples from batch B. Inter-
sugar values significantly higher than those observed in esting differences among the batches were also found in
samples from batch B were found. Whereas, after the 3rd other parameters such as acetic, tartaric and l-malic acid
(Table 3). Specifically, the acetic acid content was very low
Table 3  Chemical analysis of the wines at the end of fermentation:
in the wines from batch A and batch B (monoculture), with
Batch A: H. guilliermondii LS77 + S. cerevisiae AM37 (sequential similar values of 0.10 g L−1 (± 0.02), compared to the wine
inoculum); Batch B: S. cerevisiae AM37 (monoculture); Batch C: from batch C that showed a value of 0.80 g L ­ −1 (± 0.09).
spontaneous fermentation Furthermore, tartaric acid levels detected in batch A were
Wines lower than those registered in wines from batches B and C.
While the wine from batch A showed a level in l-malic acid
Batch A Batch B Batch C
considerably higher than that found in the other batches.
pH 2.95 ± 0.11a* 2.85 ± 0.10b 2.93 ± 0.10a
Total acidity (g L−1) 7.02 ± 0.73a 7.64 ± 0.74b 8.33 ± 0.79c Volatile compounds
Reducing sugar (g L−1) 0.15 ± 0.03a 0.15 ± 0.03a 4.60 ± 0.18b
Alcohol (vol%) 12.50 ± 0.73a 12.40 ± 0.73b 11.87 ± 0.71c The concentration of the volatile compounds assayed to
Dry extract (g L−1) 19.60 ± 1.83a 20.00 ± 1.93b 20.10 ± 1.94b describe the compositional changes that occurred in the
Total ash (g L−1) 1.38 ± 0.20a 1.14 ± 0.14b 1.20 ± 0.18b wines from the three batches (A, B and C) is reported in
Alkalinity ashes 15.40 ± 0.74a 15.20 ± 0.73a 18.80 ± 0.83b Table 4.
(meq L−1) In the wines from batches A (sequential inoculum) and C
Total phenols (mg L−1) 244.00 ± 42a 240.00 ± 40a 260.00 ± 53b (spontaneous fermentation), a higher amount of terpenes was
Glycerol (g L−1) 8.30 ± 0.83a 5.70 ± 0.63b 8.20 ± 0.82a found. Specifically, linalool had values of 1136.2 mg L−1
Acetic acid (g L−1) 0.10 ± 0.02a 0.10 ± 0.02a 0.80 ± 0.09b (± 116.1) in batch A and 1338.1 mg L−1 (± 80.6) in batch
Tartaric acid (g L−1) 17.40 ± 1.73a 20.50 ± 1.93b 22.40 ± 2.03c C, while the wine from batch B showed a low linalool con-
−1
l-Malic acid (g L ) 1.20 ± 0.18a 1.40 ± 0.24b 1.22 ± 0.19a tent (770.2 mg L−1 ± 90.6). Other compounds that showed
−1
l-Lactic acid (g L ) nd nd nd meaningful differences among the batches were higher
−1
d-Gluconic acid (g L ) 0.10 ± 0.02a 0.10 ± 0.02a 0.10 ± 0.02a alcohol contents. In particular, 2-3-butanediol showed
−1
Catechins (mg L ) 18.80 ± 0.83a 21.70 ± 1.66b 18.20 ± 0.80a concentrations of 543.4 mg L−1 (± 55.4), 356.1 mg L−1
Data are means ± standard deviations of three separate replicates (± 10.1) and 468.2 mg L−1 (± 24.3), in the wines of batches
*a–c: within a row, different letters indicate significant differences A, B and C, respectively. In the case of 2-methyl-butanol,
(P ≤ 0.05) higher levels were detected in the wines from batches A

Table 4  Volatile compounds Volatile compounds (mg L−1) Wines

of wines at the end of
alcoholic fermentation in Batch A Batch B Batch C
sequential inoculum in
batch A: H. guilliermondii 2-Phenylethanol 149.1 ± 7.7a* 82.2 ± 8.4b 42.5 ± 1.2c
LS77 + S. cerevisiae AM37; 2-3-Butanediol 543.4 ± 55.4a 356.1 ± 10.1b 468.2 ± 24.3c
batch B: S. cerevisiae AM37 Methanol 38.3 ± 3.9a 27.6 ± 1.4b 45.2 ± 1.2c
in monoculture; batch C:
spontaneous fermentation Propan-1-ol 23.1 ± 2.4a 49.2 ± 2.55b 29.3 ± 0.8c
2-Methyl-propan-1-ol 60.2 ± 6.2a 42.3 ± 2.2b 21.2 ± 0.6c
3-Methyl-butan-1-ol nd 69.1 ± 3.6 nd
2-Methyl-butanol 292.4 ± 30.1a 247.4 ± 12.8b 206.3 ± 5.8c
1-Octen-3-ol 237.6 ± 24.3a 177.3 ± 9.2b 271.1 ± 7.6c
Linalool 1136.2 ± 116.1a 770.2 ± 90.6b 1338.1 ± 80.6c
Terpinen-4-ol 82.1 ± 8.4a 109.4 ± 5.8b 43.4 ± 1.2c
Nonanol nd 12.3 ± 0.1 nd
a-Terpineol nd nd 12.1 ± 0.3
Nerol 20.7 ± 2.1a 17.5 ± 0.2b 12.2 ± 0.3c

Data are means ± standard deviations of three separate replicates

nd not detected
*a–c: within a row, different letters indicate significant differences (P ≤ 0.05)

13
World Journal of Microbiology and Biotechnology (2018) 34:161 Page 7 of 10  161
and B 292.4 mg L−1 (± 30.1) and 247.4 mg L−1 (± 12.8)
respectively. In batch C, the content of 2-methyl-butanol,
was 206.3 mg L−1 (± 5.8). Significantly higher values of
1-octen-3-ol were also detected in the wine of batch C
(271.1  mg  L−1 ± 7.6) than in the wines from batches A
and B 237.6 mg L−1 (± 24.3) and 177.3 mg L ­ −1 (± 9.2),
respectively.
Sensory profile of wines
The test allowed to evaluate significant differences in the
sensory properties among the wines obtained by spontane-
ous fermentation as well as those obtained by adding the
yeast starter cultures (sequential inoculum and monocul-
ture). From Fig. 1, it is evident that wine obtained with the
sequential inoculum (batch A) showed the highest scores for
aromatic intensity (4.4).
The average score obtained for the three different wines
in their floral notes and fruitiness, did not have significant
differences among them. Moreover, the wines obtained by
fermentation with the sequential inoculum (batch A) and
those obtained with the monoculture (batch B), had the high-
est scores regarding their softness and overall evaluation.
Finally, the sensory analysis of the wine obtained by the
spontaneous fermentation (batch C) revealed the presence of
high values of acidity, with a negative impact in the sensory
perception.
Fig. 1  Sensory profiles of Cam-
panino white wine produced on
an industrial scale using H. guil-
liermondii LS77 + S. cerevisiae
AM37 in sequential inoculum
(batch A); S. cerevisiae AM37
in monoculture (batch B); spon-
taneous fermentation (batch C).
The data represent the means
sensory scores of three replicate
fermentations. a–c: different
letters indicate significant differ-
ences (P ≤ 0.05). *Emphasizes
the descriptors under which at
least one wine was perceived
differently from others
Discussion
This work presents new findings about the fermentation
behavior of the sequential inoculum of H. guilliermondii
and S. cerevisiae in the production of traditional white wine,
removing relevant doubts relating to the use of apiculate
yeast in winemaking.
So far, several Authors (Ciani et al. 2010; Jolly et al.
2014; Lleixà et al. 2016; Tristezza et al. 2016) have stud-
ied the addition of non-Saccharomyces yeast in mixture or
in sequential inoculum with S. cerevisiae, highlighting that
the use of non-Saccharomyces represents a biotechnological
strategy to improve wine quality. More in depth, the use of
non-Saccharomyces affects both the primary and the sec-
ondary aroma of wines through the production of enzymes
and metabolites, respectively. Padilla et al. (2016) reported
that specific non-Saccharomyces yeasts combined with S.
cerevisiae represents a feasible alternative to spontaneous
or inoculated fermentations and that precise combinations
could be used to produce wines with unique aromatic char-
acteristics that reflect the characteristic of a specific wine
production region. Interestingly, some studies (Rojas et al.
2003; Moreira et al. 2008) have reported that a desirable
characteristic such as the increase of fruity acetate esters has
been the main target of mixed starters designed with Hanse-
niaspora species. However, the interaction between Saccha-
romyces and non-Saccharomyces yeast remains unclear and
the effects of Hanseniaspora spp. on alcoholic fermentation
13
 161   Page 8 of 10
is doubtful as starter in industrial scale winemaking. The
competition for nutrients or the higher consumption of
amino acids and vitamins by non-Saccharomyces yeast could
negatively affect the growth of Saccharomyces spp. Moreo-
ver, the production of different metabolites can vary depend-
ing on the fermentation scale (laboratory, pilot or industrial)
and the oxygen conditions (Viana et al. 2011) In our study,
for the first time the effect of a specific H. guilliermondii
strain on winemaking of traditional white wine (Campanino)
through an industrial scale has been investigated.
Results have taken away any doubt on the use of H. guil-
liermondii in winemaking, evidencing a positive effect of
sequential inoculum on the alcoholic fermentation behav-
iour. In fact, the presence of the H. guilliermondii strain
from the early stages of fermentation seems to interact posi-
tively with S. cerevisiae and it affects the main oenological
parameters enhancing the final quality. As evidenced for
other kinds of food (Tremonte et al. 2007, 2010), interac-
tions between useful microorganisms intentionally added
could positively influence some features of technological
interest. Specifically, in this study, the highest content of
glycerol in wine obtained by the use of sequential inoculum
suggests that glycerol production could be related to cell
concentration and persistence of Hanseniaspora spp. This
effect enhances the sensorial quality of the end-product,
since the glycerol contributes to the smoothness (mouth-
feel), sweetness and complexity in wines (Lombardi et al.
2015). The positive effects produced by H. guilliermondii
were also emphasized by the results from the investigation
of volatile compounds. The presence and the persistence
of wild non-Saccharomyces or intentionally added H. guil-
liermondii strain affect the sensorial characteristics of wine
by increasing the production of β-glucosidase activity and
consequentially the amounts of volatile terpenes and higher
alcohol contents. Furthermore, the results have clearly indi-
cated that H. guilliermondii in sequential inoculum with S.
cerevisiae improved the formation 2-phenylethanol, that as
reported in literature (Medina et al. 2013; Tristezza et al.
2016) produce a positive sensorial impact. These effects
were confirmed also by sensorial analyses indicating the
highest values in aromatic intensity, persistence, softness,
floral notes and fruitiness for the wine obtained with sequen-
tial inoculum. In addition, H. guilliermondii produces wines
with lower acetic acid content than those detected in wines
obtained by spontaneous fermentation.
At the best of our knowledge, for the first time the effect
of an autochthonous and well selected non-Saccharomyces
strain on the overall quality of a typical white wine (Cam-
panino) was highlighted trough an industrial scale. There-
fore, the sequential inoculum of H. guilliermondii and
Saccharomyces yeasts represent a feasible tool to enhance
quality of traditional wine that indicate the characteristic of
a specific wine production region.
13
World Journal of Microbiology and Biotechnology (2018) 34:161
Acknowledgements  Research in our labs is funded by Grants
AGL2015-64522-C2-R (Spanish Ministry of Economy and Com-
petitiveness), ALIBIRD-CM 2013 S2013/ABI-2728 (Comunidad de
Madrid) and within the framework of the project by Molise Region
(Rural Development Programme 2007–2013; Measure 1.2.4. Coopera-
tion for development of new products, processes and technologies in
the agriculture and food sector and in forestry).
Compliance with ethical standards 
Conflict of interest The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that
could be interpreted as a potential conflict of interest.
References
Andorrà I, Berradre M, Rozès N, Mas A, Guillamón JM, Esteve-Zar-
zoso B (2010) Effect of pure and mixed cultures of the main wine
yeast species on grape must fermentations. Eur Food Res Technol
231(2):215–224
Belda I, Ruiz J, Alastruey-Izquierdo A, Navascués E, Marquina D,
Santos A (2016) Unraveling the enzymatic basis of wine “flavo-
rome”: a phylo-functional study of wine related yeast species.
Front Microbiol 7:12
Belda I, Ruiz J, Esteban-Fernández A, Navascués E, Marquina D,
Santos A, Moreno-Arribas MV (2017) Microbial contribution to
wine aroma and its intended use for wine quality improvement.
Molecules 22(2):189
Brizuela NS, Bravo-Ferrada BM, Valdés La Hens D, Hollmann A,
Delfederico L, Caballero A, Tymczyszyn EE, Semorile L (2017)
Comparative vinification assays with selected Patagonian strains
of Oenococcus oeni and Lactobacillus plantarum. LWT Food Sci
Technol 77:348–355
Cavazza A, Grando MS, Zini C (1992) Rivelazione della microflora
microbica di mosti e vini. Vignevini 9:17–20
Ciani M, Comitini F, Mannazzu I, Domizio P (2010) Controlled mixed
culture fermentation: a new perspective on the use of non-Saccha-
romyces yeasts in winemaking. FEMS Yeast Res 10(2):123–133
Ciani M, Capece A, Comitini F, Canonico L, Siesto G, Romano P
(2016) Yeast interactions in inoculated wine fermentation. Front
Microbiol 7:555
Cocolin L, Pepe V, Comitini F, Comi G, Ciani M (2004) Enological
and genetic traits of Saccharomyces cerevisiae isolated from for-
mer and modern wineries. FEMS Yeast Res 5(3):237–245
Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I,
Ciani M (2011) Selected non-Saccharomyces wine yeasts in con-
trolled multistarter fermentations with Saccharomyces cerevisiae.
Food Microbiol 28:873–882
De Leonardis A, Macciola V, Iorizzo M, Lombardi SJ, Lopez F, Mar-
coni E (2018) Effective assay for olive vinegar production from
olive oil mill wastewaters. Food Chem 240:437–440
European Commission (1990) Commission Regulation (EC) No
2676/90 determining community methods for the analysis of
wines. Off J Eur Comm L272:1–192
Ferraro L, Fatichenti F, Ciani M (2000) Pilot scale vinification process
using immobilized Candida stellata cells and Saccharomyces cer-
evisiae. Process Biochem 35(10):1125–1129
Ferreira AM, Climaco MC, Faria AM (2001) The role of non-Sac-
charomyces species in releasing glycosidic bound fraction of
grape aroma components e a preliminary study. J Appl Microbiol
91:67–71
Fleet GH (2008) Wine yeasts for the future. FEMS Yeast Res
8(7):979–995
World Journal of Microbiology and Biotechnology (2018) 34:161 Page 9 of 10  161
Garcìa M, Esteve-Zarzoso B, Arroyo T (2016) Non-Saccharomyces
yeast: Biotechnological role for wine production. In: Grape and
wine biotechnology. Chapter 11:250–271
Iorizzo M (1997) Caratterizzazione enologica di lieviti apiculati iso-
lati da mosti d’uva dell’Italia meridionale. Doctoral dissertations,
University of Molise
Iorizzo M, Macciola V, Testa B, Lombardi SJ, De Leonardis A (2014)
Physicochemical and sensory characteristics of red wines from
the rediscovered autochthonous Tintilia grapevine grown in the
Molise region (Italy). Eur Food Res Technol 238(6):1037–1048
Iorizzo M, Testa B, Lombardi SJ, García-Ruiz A, Munoz-Gonzalez C,
Bartolome B, Moreno-Arribas MV (2016) Selection and techno-
logical potential of Lactobacillus plantarum bacteria suitable for
wine malolactic fermentation and grape aroma release. Food Sci
Technol 73:557–566
Jolly NP, Varela C, Pretorius IS (2014) Not your ordinary yeast: non-
Saccharomyces yeasts in wine production uncovered. FEMS Yeast
Res 14(2):215–237
Liu PT, Lu L, Duan CQ, Yan GL (2016) The contribution of indigenous
non-Saccharomyces wine yeast to improved aromatic quality of
Cabernet Sauvignon wines by spontaneous fermentation. LWT
Food Sci Technol 71:356–363
Lleixà J, Martin V, del C Portillo, Carrau M, Beltran F, Mas G A (2016)
Comparison of fermentation and wines produced by inoculation
of Hanseniaspora vineae and Saccharomyces cerevisiae. Front
Microbiol 7:338
Lombardi SJ, De Leonardis A, Lustrato G, Testa B, Iorizzo M (2015)
Yeast autolysis in sparkling wine aging: use of killer and sensi-
tive Saccharomyces cerevisiae strains in co-culture. Recent Pat
Biotechnol 9:1–8
Medina K, Boido E, Fariña L, Gioia O, Gomez ME, Barquet M, Car-
rau F (2013) Increased flavour diversity of Chardonnay wines by
spontaneous fermentation and co-fermentation with Hansenias-
pora vineae. Food Chem 141(3):2513–2521
Mendoza L, Farías ME (2010) Improvement of wine organoleptic char-
acteristics by non-Saccharomyces yeasts. Curr Res Technol Educ
Topics Appl Microbiol Microb Biotechnol 2:908–919
Moreira N, Mendes F, Guedes de Pinho P, Hogg T, Vasconcelos I
(2008) Heavy sulphur compounds, higher alcohols and esters
production profile of Hanseniaspora uvarum and Hanseniaspora
guilliermondii grown as pure and mixed cultures in grape must.
Int J Food Microbiol 124:231–238
Moschetti G, Corona O, Gaglio R, Squadrito M, Parrinello A, Settanni
L, Barone E, Francesca N (2016) Use of fortified pied de cuve as
an innovative method to start spontaneous alcoholic fermentation
for red winemaking. Aust J Grape Wine Res 22(1):36–45
Padilla B, Gil JV, Manzanares P (2016) Past and future of non-Saccha-
romyces yeast: from spoilage microorganisms to biotechnologi-
cal tools for improving wine aroma complexity. Front Microbiol
7:411
Pallmann CL, Brown JA, Olineka TL, Cocolin L, Mills DA, Bisson LF
(2001) Use of WL medium to profile native flora fermentations.
Am J Enol Viticult 52:198–203
Reale A, Di Renzo T, Succi M, Tremonte P, Coppola R, Sor-
rentino E (2013) Microbiological and fer mentative
properties of baker’s yeast starter used in breadmaking. J Food
Sci 78(8):M1224–M1231
Rojas V, Gil JV, Piñaga F, Manzanares P (2003) Acetate ester formation
in wine by mixed cultures in laboratory fermentations. Int J Food
Microbiol 86:181–188
Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of
total phenols and other oxidation substrates and antioxidants by
means of Folin–Ciocalteu reagent. Method Enzymol 299:152–178
Succi M, Pannella G, Tremonte P, Tipaldi L, Coppola R, Iorizzo M,
Lombardi SJ, Sorrentino E (2017a) Sub-optimal pH preadaptation
improves the survival of Lactobacillus plantarum strains and the
malic acid consumption in wine-like medium. Front Microbiol
8:470
Succi M, Tremonte P, Pannella G, Tipaldi L, Cozzolino A, Coppola
R, Sorrentino E (2017b) Survival of commercial probiotic strains
in dark chocolate with high cocoa and phenols content during
the storage and in a static in vitro digestion model. J Funct Foods
35:60–67
Testa B, Lombardi SJ, Tremonte P, Succi M, Tipaldi L, Pannella G,
Sorrentino E, Iorizzo M, Coppola R (2014) Biodiversity of Lacto-
bacillus plantarum from traditional Italian wines. World J Micro-
biol Biotechnol 30(8):2299–2305
Tremonte P, Succi M, Reale A, Di Renzo T, Sorrentino E, Coppola R
(2007) Interactions between strains of Staphylococcus xylosus and
Kocuria varians isolated from fermented meats. J Appl Microbiol
103(3):743–751
Tremonte P, Reale A, Di Renzo T, Tipaldi L, Di Luccia A, Coppola R,
Sorrentino E, Succi M (2010) Interactions between Lactobacillus
sakei and CNC (Staphylococcus xylosus and Kocuria varians)
and their influence on proteolytic activity. Lett Appl Microbiol
51(5):586–594
Tremonte P, Pannella G, Succi M, Tipaldi L, Sturchio M, Coppola R,
Luongo D, Sorrentino E (2017a) Antimicrobial activity of Lacto-
bacillus plantarum strains isolated from different environments:
a preliminary study. Int Food Res J 24(2):852–859
Tremonte P, Sorrentino E, Pannella G, Tipaldi L, Sturchio M, Masucci
A, Maiuro L, Coppola R, Succi M (2017b) Detection of differ-
ent microenvironments and Lactobacillus sakei biotypes in Ven-
tricina, a traditional fermented sausage from central Italy. Int J
Food Microbiol 242:132–140
Tristezza M, Tufariello M, Capozzi V, Spano G, Mita G, Grieco F
(2016) The oenological potential of Hanseniaspora uvarum in
simultaneous and sequential co-fermentation with Saccharomyces
cerevisiae for industrial wine production. Front Microbiol 7:670
Viana F, Belloch C, Vallés S, Manzanares P (2011) Monitoring a mixed
starter of Hanseniaspora vineae–Saccharomyces cerevisiae in
natural must: impact on 2-phenylethyl acetate production. Int J
Food Microbiol 151:235–240
Wang C, Mas A, Esteve-Zarzoso B (2016) The interaction between
Saccharomyces cerevisiae and non-Saccharomyces yeast during
alcoholic fermentation is species and strain specific. Front Micro-
biol 7:502
Zohre DE, Erten H (2002) The influence of Kloeckera apiculata and
Candida pulcherrima yeasts on wine fermentation. Process Bio-
chem 38(3):319–324
13
 161   Page 10 of 10 World Journal of Microbiology and Biotechnology (2018) 34:161

Affiliations

Silvia Jane Lombardi1 · Gianfranco Pannella1 · Massimo Iorizzo1   · Maria Victoria Moreno‑Arribas2 ·

Patrizio Tremonte1 · Mariantonietta Succi1 · Elena Sorrentino1 · Vincenzo Macciola1 · Massimo Di Renzo1,3 ·
Raffaele Coppola1

* Massimo Iorizzo Massimo Di Renzo

iorizzo@unimol.it massimo.direnzo@mastroberardino.com
Silvia Jane Lombardi Raffaele Coppola
silvia.lombardi@unimol.it coppola@unimol.it
Gianfranco Pannella 1
Department of Agriculture, Environmental and Food
gianfranco.pannella@unimol.it
Sciences, University of Molise, Campobasso, CB, Italy
Maria Victoria Moreno‑Arribas 2
Instituto de Investigación en Ciencias de la Alimentación
victoria.moreno@csic.es
(CIAL), CSIC-UAM, C/Nicolás Cabrera 9. Campus de
Patrizio Tremonte Cantoblanco, CEI UAM+CSIC, 28049 Madrid, Spain
tremonte@unimol.it 3
Mastroberardino spa winery, via Manfredi, 75‑81,
Mariantonietta Succi 83042 Atripalda, AV, Italy
succi@unimol.it
Vincenzo Macciola
macciola@unimol.it

13

View publication stats