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Impact of the Physicochemical Composition and Microbial Diversity in Apple

Juice Fermentation Process: A Review

Article in Molecules · August 2020

DOI: 10.3390/molecules25163698

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Review

Impact of the Physicochemical Composition and

Microbial Diversity in Apple Juice Fermentation
Process: A Review
Marina Al Daccache 1,2, Mohamed Koubaa 3,*, Richard G. Maroun 2, Dominique Salameh 2,
Nicolas Louka 2 and Eugène Vorobiev 1
1 Sorbonne University, Université de technologie de Compiègne, ESCOM, EA 4297 TIMR, Centre de
recherche Royallieu, CS 60319, 60203 Compiègne CEDEX, France; [email protected] (M.A.D.);
[email protected] (E.V.)
2 Laboratoire CTA, UR TVA, Centre d’Analyses et de Recherche, Faculté des Sciences, Université

Saint‐Joseph, Beyrouth 1104 2020, Lebanon; [email protected] (R.G.M.);

[email protected] (D.S.); [email protected] (N.L.)
3 ESCOM, UTC, EA 4297 TIMR, 1 allée du réseau Jean‐Marie Buckmaster, 60200 Compiègne, France

* Correspondence: [email protected]; Tel.: +33‐3442‐38841

Academic Editor: Krystian Marszałek

Received: 10 July 2020; Accepted: 11 August 2020; Published: 13 August 2020

Abstract: Fermented apple beverages are produced all over the world with diverse characteristics
associated with each country. Despite the diversifications, cider producers are confronted with
similar issues and risks. The nature of the raw material, also known as the fermentation medium,
plays a key role in fermentation. A well‐defined composition of apples is, therefore, required to
produce cider with good quality. In addition, ferment and its metabolism are important factors in
the fermentation process. The producers of cider and other alcoholic beverages are looking in
general for novel yeast strains or for the use of native strains to produce “authentic” and diversified
beverages that are distinct from each other, and that attract more and more consumers. Research
articles on cider production are infrequent compared to wine production, especially on the impact
of the chemical composition and microbial diversity of apples on fermentation. Even though the
processing of fermented beverages is close in terms of microbial interactions and production, the
study of the specific properties of apples and the production challenges of cider production is
advantageous and meaningful for cider producers. This review summarizes the current knowledge
on apple composition and the impact of the must composition on fermentation and yeast growth.
In addition, the microbial diversity of cider, activities, and its influence on fermentation are
reviewed.

Keywords: apples; cider; fermentation; physicochemical composition; microorganisms

1. Introduction
Apples represent a very particular fruit known for their unique symbolic richness over time.
Later, the different studies proved the importance of that fruit due to its chemical composition and
specifically its antioxidant characteristics. The fruit belongs to the “Maloideae” subfamily and to the
“Rosacea” family. It represents one of the most important deciduous tree fruits that are generally
grown in temperate and tropical regions [1]. Apple is one of the most produced and consumed fruits
in the world. It is ranked as the third‐most fruit produced worldwide after bananas and watermelon
with a production that reached 75 million tons in 2018–2019 [2]. China stands as the largest producer

Molecules 2020, 25, 3698; doi:10.3390/molecules25163698 www.mdpi.com/journal/molecules

Molecules 2020, 25, 3698 2 of 16

with increasing production of almost 33 million tons per year, followed by the European Union (EU)
producing 15 million tons per year (Table 1). The United States comes in the third rank, producing
5.6 million tons of apples in 2019. The main apple producers in Europe are Poland, France, and Italy.
Turkey and Iran produce around 3 million tons per year each while the production of Chile, Russia,
Ukraine, and Brazil is around 1.2 million tons per year (Table 1).

Table 1. Global production and consumption of fresh apples per year in 2019 [2].

Fresh Domestic
Country Apple Production (kt)
Consumption (kt)
China 33,000 38,050
European Union 15,442 7400.6
United States 5564 2589.4
Turkey 3306 2630.5
Iran 3085 1813.9
Russia 1656 1884.4
Chile 1393 229.6
Ukraine 1211 1066.2
Brazil 1156 1325.9

Furthermore, apple juice is the main raw material for several beverages’ production. Vinegar,
cider, calvados, and apple wine are obtained from apple juice fermentation, depending on the
conditions applied. This review focuses on the alcoholic fermentation of apple juice to produce cider.
Over the past years, different definitions were accorded to the word “fermentation”. The term was
first applied to describe the production of wine and specifically the bubbling caused by the
production of carbon dioxide. Nowadays, alcoholic fermentation is known as a biological complex
process where yeasts convert sugars like glucose, fructose, and sucrose into cellular energy, ethanol,
carbon dioxide, and other metabolic byproducts. Different parameters may affect the fermented
product such as the composition of the raw materials, the microorganism used during the
fermentation, and the process parameters and conditions.

2. Physicochemical Composition and Microbial Diversity of Apple Fruits

2.1. Fresh Apple Composition

Mature apples are mainly composed of about 85% water, 12–14% carbohydrates, 0.30–1%
organic acid, 0.30% proteins, and less than 0.10% lipids, minerals, and vitamins [3]. The variation in
the biochemical composition is mainly related to the variety and maturity, as well as the agronomic
and climatic conditions. Furthermore, the soil composition may have a great impact on the fruit
quality and composition. For example, nitrogen has a significant impact on fruit size and
composition. High levels of nitrogen in apples may reduce the amount of soluble solids and raise the
titratable acidity [4]. In addition, potassium intensifies the fruit color and significantly decreases the
fruit firmness. Enough potassium concentration increases the apple sugar content, improves the fruit
quality, the surface color, aroma, flavor, and storability [5]. Furthermore, calcium can be positively
associated with fruit firmness and prevents the fruit from softening during storage [6,7]. Boron is
also an essential trace element for the normal growth and development of apples.

2.1.1. Sugars
The two main monosaccharides in apples are glucose and fructose, whereas sucrose is the main
disaccharide (Table 2). D‐sorbitol, in the form of alcoholic sugar, is abundant in apples with a
concentration of 300 to 800 mg/100 mL of apple juice [8]. In addition, all fruits contain cellulose,
hemicellulose (pentosans), and pectin.
Molecules 2020, 25, 3698 3 of 16

Table 2. Simple and parietal sugar contents of apple fruit [8,9].

Compounds Concentrations
Glucose * 1.8
Fructose * 5.6
Sucrose * 2.6
Arabinose ** 109
Rhamnose ** 12
Fucose ** 8.5
Galactose ** 71.5
Glucose ** 288
Mannose ** 21
Xylose ** 57
Galacturonic Acid ** 227
*g/100 g of fresh fruit; **mg/g of the cell wall materials.

2.1.2. Organic Acids

L‐malic acid is the main organic acid found in apples. Citric acid is found in very low
concentrations, with thousands of times lower concentration than that of malic acid. The apple
contains at least nine to 12 acids in small fractions. The acidity of apples can vary from 0.1 to 2.5 g of
malic acid per 100 g of juice [10]. In addition to malic acid, citric, succinic, citramalic, shikimic,
glyceric, glyoxylic, isocitric, glycolic, lactic, and galacturonic acids are present in apple juice. Three
different keto acids have been found: Oxaloacetic acid, pyruvic acid, and ketoglutaric acid. However,
these acids represent only a small fraction of the total organic acids present.

2.1.3. Phenolic Compounds

The measurement of the phenolic substances present in the fruit has been of great interest to
pomologists and cider producers (Table 3). Phenolic compounds, which are secondary plant
metabolites, play a major role in the sensory, nutritional, and antioxidant properties of the fruit.
Structurally, polyphenols are all composed of one or more aromatic rings with different structures
that allow their classification into categories. Phenolic compounds are distinguished by the number
and sequence of aromatic rings, the number and position of hydroxyl groups, and the presence of
nonphenolic substitutes like, for example, alkyl groups, sugars, and organic acids. The phenolic
compounds found in apples fall into two categories: Phenolic acids and flavonoids.

Table 3. Average content of phenolic compounds in apple flesh and peel (single compounds and total concentrations
are expressed in mg/100 g freeze‐dried material, TPC denotes total phenolic compounds and is expressed in mg
GAE/100 g freeze‐dried material) [11]. “n.d.” denotes “not detected”, “GAE” denotes “Gallic Acid Equivalent”.

Compounds Apple Flesh Apple Peel

Procyanidin B1 1.0 ± 0.1 1.9 ± 1.6
(+) ‐catechin 1.1 ± 0.9 5.1 ± 3.0
Procyanidin B2 1.6 ± 0.3 7.1 ± 1.5
Procyanidin C1 n.d. 6.7 ± 1.2
(−) ‐epicatechin 1.0 ± 0.7 8.8 ± 4.9
Procyanidin A2 2.5 ± 1.2 7.8 ± 2.9
Total Flavanols 6.4 ± 2.5 36.1 ± 8.8
Gallic acid 1.6 ± 0.2
Protocatechuic acid 0.1 ± 0.0 1.3 ± 0.6
Chlorogenic acid 2.1 ± 0.8 5.6 ± 0.7
Caffeic acid 0.8 ± 0.5 0.8 ± 0.5
P‐coumaric acid 0.5 ± 0.1 1.8 ± 0.4
Ferulic acid 0.1 ± 0.1 0.9 ± 0.4
Molecules 2020, 25, 3698 4 of 16

Total Phenolic Acids 5.2 ± 1.3 14.3 ± 2.1

Phloridzin 1.1 ± 0.7 4.8 ± 3.5
Hyperoside n.d. 84.2 ± 57.1
Isoquercitrin n.d. 16.6 ± 8.6
Rutin n.d. 5.4 ± 3.3
Reynoutrin n.d. 17.0 ± 7.6
Avicularin n.d. 21.4 ± 8.4
Quercitrin n.d. 25.4 ± 13.1
Quercetin n.d. 13.4 ± 5.2
Total flavonols n.d. 183.5 ± 99.6
Total polyphenols 12.6 ± 4.4 239.4± 118.6
TPC 179.5 ± 52.3 914.7± 331.3

Phenolic Acids
Hydroxycinnamic acid esters represent one of the main classes of phenolic acids in different
apple varieties [12]. Hydroxycinnamic acid is found in its esterified form. Chlorogenic acid, an ester
of caffeic and quinic acid, is present in relatively high concentrations in both the peel and flesh of
most apple cultivars. Chlorogenic acid oxidation is mainly responsible for the browning occurring in
apple juice and cider [13]. P‐coumaroylquinic acid is also found in apple fruit, but in lower
concentrations [12].

Flavonoids
Flavonoids are widely found in different fruit and vegetable tissues such as leaves, seeds, bark,
and flowers [14]. Flavonoids’ skeleton is composed of diphenylpropane (C6‐C3‐C6) with the
three‐carbon bridge between the phenyl groups connected with oxygen [15]. Thirteen subgroups of
flavonoids exist according to differences in the number of substituted hydroxyl groups, degree of
unsaturation, and degree of oxidation of the three‐carbon bridge [15,16]. Flavonoids are referred to
as glycosides when they contain one or more sugars and as aglycones when no sugar group is
present [17]. In apple, anthocyanidin, flavanol (also named flavan‐3‐ols), flavonols (mainly quercetin
glycosides), and dihydrochalcones are the major subgroups of flavonoids [18,19].

Flavan‐3‐ols
Flavan‐3‐ols’ class is the only class of flavonoids found in plants under their aglycone form. The
skeleton of the base unit is represented by the flavane nucleus. It is a very large family, constituted of
11 subclasses. Flavan‐3‐ols exist in the monomeric or polymeric form. The monomeric flavan‐3‐ols
constitute the second‐largest class of polyphenols in apple fruit after hydroxycinnamic acids. They
are represented only by (+)‐catechin (CAT) and (‐)‐epicatechin (EC). The (‐)‐epicatechin is always
predominantly present in apples with concentrations ranging from 46 mg.kg−1 to 2225 mg.kg−1 in
Golden Delicious. The (+)‐catechin is a minor flavanol, of which the concentrations may vary from 6
mg.kg−1 in Broxwood Foxwhelp to 408 mg.kg−1 in Yarlington Mill in the flesh [19,20].

Flavonols
Flavonols are commonly found in all fruits as 3‐glycosides. Quercetin, an efficient antioxidant,
is found in high concentrations of 49 mg/kg of apples [8]. Flavonols are usually yellow compounds
that are responsible for the yellow color of certain apples’ epidermis.

Dihydrochalcone
Dihydrochalcones represent a specific class of flavonoids found in apple fruits. The aglycone
dihydrochalcone is phloretin found only in a glycoside form in the fruit. Phloridzin (PLZ, phloretin
glucoside) and phloretin xyloglucoside (XPLT) are the most widely described glycosides in the
Molecules 2020, 25, 3698 5 of 16

literature [19,21]. This family is particularly concentrated in pips. They can thereby represent more
than 3 g/kg, i.e., 66% of the present phenolic compounds [19].

2.1.4. Lipids
Apple fruits have commonly low lipid content, ranging from 0.1 to 0.5% of fresh weight. High
levels of lipids are usually detected in the fruit seeds. The lipid fraction in fruits is composed of
triacylglycerols, glycolipids and phospholipids, carotenoids, triterpenoids, and waxes. The lipid
content in the apple fruits is detailed in Table 4.

Table 4. Lipids content of apple flesh [8].

Compounds % of Total Lipids

Triacylglycerols 5
Glycolipids 17
Phospholipids 47
Sterols 15
Sterol esters 2
Sulfolipids 1
Others 13

2.1.5. Vitamins
Vitamin C is biosynthesized in plants from hexoses such as glucose. Vitamin C content, also
known as L‐ascorbic acid, is ranging from 3 to 35 mg/100 g of the edible portion of the apple. It has a
very high antioxidant activity. Vitamin B12, vitamin D, and tocopherols are found in trace amounts
[8].

2.1.6. Minerals
Minerals, also called inorganic nutrients, are found in all fruits. The most important cation and
anion are potassium and phosphorus, respectively. Other elements such as sodium, calcium, and
iron are also present at lower concentrations (Table 5).

Table 5. Mineral contents in apple [8].

Minerals mg/100 g Dry Matter

Potassium 840
Sodium 7.9
Calcium 38
Magnesium 40
Iron 1.6
Aluminum 0.43
Phosphorus 73
Zinc 0.65
Manganese 0.3
Copper 0.35

2.2. Microbial Ecology of Apple Fruit and Cider

Fruits are characterized by a predominant microflora that varies from a fruit to another and
may be affected by the environment, the geographical area, the weather conditions, the pesticides, or
the conditions under which the fruit was developed [22–24]. For cider, the perception of
“territoriality” is necessary for its appreciation. Its sensory profile is expressively related to microbial
activities, and indigenous microorganisms may dynamically contribute to the expression of cider
authenticity. Ciders’ microbial ecology comprises numerous genera, species, and strains of bacteria
and yeasts. A few studies described the microbiological diversity of fresh apple fruit, apple juice,
Molecules 2020, 25, 3698 6 of 16

and cider. Teixidó et al. [25] found that the main microbiota of “Golden Delicious” fresh apples were
fungi, such as Cladosporium and Alternaria, and yeasts. Later, Abadias et al. [26] investigated the
same apple’s variety throughout the production and identified low levels of bacteria of the family
Enterobateriaceae (Pantoea, Citrobacter, Enterobacter, Klebsiella, and Escherichia). Numerous genera of
yeasts such as Candida, Cryptococcus, Debaryomyces, Kloeckera, Kluyveromyces, Pichia, Rhodotorula,
Saccharomyces, and Zygosaccharomyces could be also present in fresh fruits [27,28]. Graça et al. [29]
found that fungi (yeasts and molds) ranged from 3.6 to 7.1 log CFU/g (colony‐forming unit/g) of
fresh‐cut apples. Most of the isolates obtained in their study were from strains of Candida sake and
Pichia fermentans. Hanseniaspora spp., Candida spp., Meyerozyma guilliermondii, Metschnikowia
pulcherrima, Cryptococcus spp., and Cystofilobasidium infirmominiatum were found at lower
percentages. Furthermore, mesophilic and psychrotrophic microorganisms were found on fresh
apples at a range of 2 to 8.9 and 1.7 to 8.4 log CFU/g, respectively [29,30]. Bacteria were also found on
fresh apples in high numbers. Lactic acid bacteria were detected at a range of 1.7–8.7 log CFU/g
[29,30]. However, a low number of acid‐tolerant bacteria, usually Acetomonas species, is frequently
present [31].
When it comes to spontaneous cider fermentation, several research studies have shown that the
genus Saccharomyces is usually predominant. The non‐Saccharomyces genera, such as Kloeckera,
Candida, Pichia, Hansenula, Hanseniaspora, and Metschnikowia, are mostly growing during the first
stages of fermentation [24]. The first group of species, which is characterized by a high metabolism
includes Saccharomyces bayanus and Saccharomyces cerevisiae [32], whereas the second one can be
classified into two categories: Apiculate yeast, having a low fermentative activity (Hanseniaspora
valbyensis, Hanseniaspora uvarum, and Hanseniaspora osmophila), and species primarily showing an
oxidative metabolism (Metschnikowia pulcherrima and Pichia guillermondii) [22,24,33]. The
non‐Saccharomyces yeasts are mainly present during the first stages of fermentation and have a low
fermentative capacity [34], while ethanol‐tolerant S. cerevisiae species are mainly detected in the
middle and final stages [35]. A novel species of Sporobolomyces sucorum sp. nov. was isolated in apple
must and was closely related to Sporobolomyces pararoseus and Sporobolomyces patagonicus [36]. Some
studies showed that the presence of Saccharomyces is not common in the must and it is related to the
surfaces and production equipment [24,37]. Suárez Valles et al. [38] justified the absence of
Saccharomyces yeasts in the must due to the usage of a fast pressing system. Moreover, Al Daccache
et al. [39] reported that Hanseniaspora sp. was the major yeast strain during spontaneous
fermentation of “Ace spur” apple juice.
Besides fungi, different genera of bacteria were detected during cider fermentation. Little is
known about microbial diversity and physiology of malolactic fermentation (MLF) in cider
production. The obligate homofermentative Lactobacillus mali [40], Lactobacillus delbrueckii subsp.
lactis [41,42], and Lactobacillus acidophilus [43] are noticeably rare. In contrast, the most frequently
found species are the heterofermentative lactobacilli: Lactobacillus collinoides [44–48], Lactobacillus
paracollinoides [49], Lactobacillus fermentum [44,50], Lactobacillus buchneri, Lactobacillus viridescens,
Lactobacillus hilgardii [44], Lactobacillus diolivorans, Lactobacillus plantarum, and Lactobacillus suebicus
[51,52]. Even though some similarities with wine have been well recognized since a long time ago
[31,53,54], Sánchez et al. [55] noted that the bacterial species and percentages are different in cider. In
their work, a culture‐based approach was used to study the diversity of lactic acid bacteria (LAB) by
molecular tools. The involved microbiota throughout the MLF were Lactobacillus, Oenococcus, and
Pediococcus. L. collinoides was present and predominant throughout the entire process, its distribution
alternated with other species such as Oenococcus oeni and Pediococcus parvulus. However, P. parvulus
cannot conduct the MLF alone and played an important role in adding flavor intensity to the final
product.

3. Cider‐Making Process
Different types of ciders exist in the market since every country has its specialty to produce
traditional ciders. French cider is usually produced following a natural process without additives or
other modern treatments, compared to the English cider. Due to the different production methods,
Molecules 2020, 25, 3698 7 of 16

French cider tends to be fruity while the English one is richer in alcohol. Even if the processes seem
to be different, many key steps are common to all of these processes (Figure 1).

Figure 1. Main steps of the cider‐making process.

Apples are first transported from the silo to be machine‐washed in water. They are sorted by
appearance to remove rotten fruits. The remaining apples are transferred for milling where they are
crushed into small pieces. In the French cider process, the apple pulp is oxidized from 30 min to up
to 5 h. The pulp is then pressed and left to settle. The fermentation step, which in France relies on
natural flora, begins with an oxidative phase. Oxygen flow is highly beneficial for this flora at the
beginning of fermentation, leading to limited growth of Saccharomyces during this step. This stage is
considered very important because this is when fruity aromas are generated. The fermentation is
conducted later by Saccharomyces for 1 to 3 months at a moderate agitation speed. As for wine
production, malolactic fermentation can occur due to bacterial growth in cider. Maturation is the
next step after fermentation when other yeasts, such as Brettanomyces anomalus can grow, which can
have a negative influence on the aromatic quality of the cider. Later, a post‐fermentation clarification
step takes place, leading to a clear product without turbidity and deposits, and which stabilizes the
cider and eliminates haziness caused by the action of proteins or tannins. This step can also eliminate
microorganisms and ensure better bacterial stability in the final product. Clarification is done either
by settling, centrifugation, or filtration. Finally, after blending and final filtration, the cider is bottled
with either carbonation or additional yeast to trigger a second fermentation in the bottle.
Some research works have been conducted to investigate the impact of power ultrasound and
pulsed electric fields (PEF) on apple juice fermentation for cider production. Ultrasound‐ and
PEF‐assisted fermentations [56,57] showed that the treatment of the yeast strain Hanseniaspora sp.,
isolated from a spontaneous fermented Lebanese “Ace Spur” apple juice [58], may contribute to
shortening the fermentation time and to reducing the ethanol content in the fermented product,
depending on the parameters applied. Further investigations are, nonetheless, required to study the
impact of these emerging technologies on the sensory properties on cider.

4. Impact of Apple Juice Composition and Microbial Diversity on Alcoholic Fermentation in the
Cider Production Process
Fermentation is a complex metabolic process when sugars are transformed into ethanol,
secondary metabolites, acids, alcohols, esters, and carbon dioxide. This transformation can be
Molecules 2020, 25, 3698 8 of 16

affected by several parameters related to the fermentation medium. Thus, the choice of apple
varieties, as well as the yeast species carrying out the fermentation process, is important (Figure 2).

Figure 2. Impact of apple varieties and yeast type on cider production.

4.1. Impact of Apple Juice Composition on Fermentation

Sugar, acids, and polyphenols represent the three major compounds that affect apple juice
fermentation [59]. Accordingly, apple selection is an important step, having a direct impact on the
quality of the final product. In countries with ancient cider traditions, special varieties of apples
known as “apple cider” are grown for their high levels of acids and phenolic compounds. However,
nowadays, dessert apples are more and more frequently used, especially in Germany, Switzerland,
and America. Consequently, in order to help cider producers to obtain an optimal mixture, acidity
ratios, polyphenols, and alcohols derived from sugars or residual sugars in their products, a
quantitative classification system was developed by Long Ashton Cider Research station in the UK
[59]. Phenolic compounds have an important effect on the sensory properties of cider such as color,
bitterness, and astringency balance, which provide the mouthfeel of cider [59,60]. The phenolic
profile may differ from one apple variety to another, but it may also depend on the year of harvest,
variety, climate, maturity, storage, and processing [61–64]. Procyanidins, composed of high
molecular compounds, play a major role in astringency, while molecules of lower weights are
responsible for bitter taste. In addition, polyphenols can influence the sweetness and acidity, thus
affecting overall aroma development during fermentation [65,66]. Not only nonvolatile phenolic
compounds play a major role during fermentation, but also the volatile phenolic compounds formed
by enzymatic reactions during fermentation contribute to the formation of the aromas of the final
product. Another factor to consider is the composition and the concentration of the initial sugars.
The nature of the sugar can also affect the fermentation process. Monosaccharides can produce
carbon dioxide faster than disaccharides. Furthermore, many other factors can play a role in the
progression of fermentation. The glucose and fructose concentrations may influence the yeast
growth, i.e., a high sugar concentration will reduce the growth rate of certain yeast strains. For sugar
concentrations between 200 and 300 g/L, a decrease in the growth rate of S. cerevisiae was observed
[67,68]. Furthermore, high sugar levels increase the yeast demand for assimilable nitrogen, which
can similarly inhibit the fermentation [69]. For low glucose concentrations, yeasts use sugars either
by respiration or fermentation. Aeration induces an increase in the biomass formed (total and per
unit of degraded sugar), and at the same time, a decrease in alcohol production and sugar
consumption; Pasteur then retained that respiration inhibits fermentation. For high concentrations
of glucose, S. cerevisiae metabolizes sugars only by fermentation. Even in the presence of oxygen,
respiration is impossible. Al Daccache et al. [39] reported different fermentative behaviors of the
Molecules 2020, 25, 3698 9 of 16

yeast Hanseniaspora sp. during the fermentation of Lebanese “Ace spur” and French “Kermerrien”
apple juices. The apples used had different chemical compositions, where the “Ace spur” apple juice
had almost the double concentrations of sugars, compared to “Kermerrien” one. Different biomass
and ethanol kinetics were obtained. In the presence of an excess of sugar, the yeast cells followed the
fermentative pathway from the first hour of fermentation. For the fermentation of “Kermerrien”
apple juice, the cells were in a respiratory mode generating biomass in the early hours of
fermentation [39]. Some variables, such as temperature and pH, can influence yeast growth rates and
the ecology and adaptation of yeast strains [67,68]. Rosend et al. [70] studied the impact of four apple
varieties grown in Estonia, Antei, Melba, Kulikovskoye, and Orlovski Sinap, on cider fermentation.
Alcoholic fermentation was carried out using the must from the apples at various stages of ripening
(i.e., unripe, ripe, overripe) and commercially available yeast strains. The differences in volatile
composition between the samples were assessed. The results showed that apple variety stands as the
principal attribute influencing the quality and aroma properties of apple cider. The maturity of the
fruit was variety‐specific, the volatile profiles of Melba variety ciders were the least affected by the
ripening stage of apples [70]. Organic acids are indicators of quality during cider fermentation. The
dominant flavor of organic acids is sourness, but they also contribute to bitterness and astringency of
cider [71]. Some yeasts can assimilate malic acid resulting in its reduction, fluctuating from 5 to 40%
[72]. When a second bacterial fermentation occurs, its level is reduced mainly by lactic acid bacteria.
During this fermentation, citric acid is transformed into acetic acid, whereas shikimic and quinic
acids are metabolized to single phenols, like catechol and ethylcatechol, and other compounds.
Organic acids may affect the yeast metabolism. The yeast enzymatic activity and the chemical
alterations are also influenced by the juice acidity [73].

4.2. Impact of Yeast on Fermentation

Yeast plays an essential role in the production of all alcoholic beverages, and the selection of an
appropriate yeast strain is crucial to control the alcohol yield and to preserve the beverage’s sensory
quality. Fermentative yeasts can use sugars anaerobically as electron donors, electron acceptors, and
carbon sources. However, the yeast action during fermentation is not only limited to the
transformation of sugars into alcohol. Yeast metabolism produces different other metabolites and
by‐products that may have an essential impact on the organoleptic quality of the fermented product
[74]. Thus, the criteria to select yeast strains for their use in fermented beverages comprise their
capability to dominate the media and to improve desired sensorial characteristics and their inability
to produce undesired compounds such as biogenic amines or off odors [75]. During spontaneous
fermentation, several yeast species may be present and could play a significant, complex, and
unpredictable role [76]. Some yeast species may be present only during the first stage of
fermentation, while others, more resistant to ethanol, are dominant during the later stages. This type
of yeast is nowadays known as belonging to the Saccharomyces strains [77]. S. cerevisiae is largely used
to produce alcoholic beverages due to its controlled and repetitive behavior as well as for the release
of its aroma precursors [78–81]. Nevertheless, fermentation is the collaboration of different species of
yeast and bacteria initially present in the product or found on the surface of the presses and
fermenters. Mixed fermentations are suggested as a feasible way to improve the complexity and
enhancing the particular and specific characteristics of the product [82]. The growth of each yeast
species is characterized by a definite metabolic activity, which determines the concentrations of
flavor compounds in the final product. Therefore, the role of non‐Saccharomyces yeasts appears
important during the fermentation process. The main yeasts present in the early stages of
fermentation belong to the genera Hanseniaspora and Candida. These species are characterized by a
low fermentation capacity and are sensitive to an alcohol concentration close to 5 or 6%. In addition,
some changes in fermentation parameters may result in the presence of yeasts such as Brettanomyces,
Kluyveromyces, Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and Saccharomycodes [83–85].
From the above‐cited yeasts, some of them may have a positive impact on fermentation by releasing
favorable aromas, but others may release undesirable aromas known as off‐flavors. Yeasts can affect
primary aroma determined by the initial composition of the product and the secondary aromas that
Molecules 2020, 25, 3698 10 of 16

are created during the fermentation, as well as the tertiary aromas generated during the maturation
of the finished product [86]. Hanseniaspora, Zygosaccharomyces, and Schizosaccharomyces pombe species
produce high amounts of volatile fatty acids, such as acetic acid [87–91], and low concentrations of
higher alcohols [92–95]. Esters and sulfur compounds are mainly produced by Candida,
Hansenisapora, Torulaspora delbrueckii, and Kazachstania gamospora [93,96–98]. Lorenzini et al. [99]
investigated the capacity of Torulaspora delbrueckii, Hanseniaspora osmophila, Hanseniaspora uvarum,
Starmerella bacillaris, and Zygosaccharomyces bailii to ferment apple juice and found that Hanseniaspora
uvarum was the greatest producer of hexyl and isoamyl acetate. The complex volatile profile of cider
suggests the possible strain‐specific effects on the aroma formation. Wei et al. [100] tried to enhance
the flavor complexity of cider by different non‐Saccharomyces species. The chemical composition and
sensory properties of five different fermentations of mixed cultures of Pichia kluyveri, Hanseniaspora
vineae, Hanseniaspora uvarum, and Torulaspora quercuum were studied for apple juice fermentation.
The results indicated that the growth of P. kluyveri and H. vineae were interreacted and affected by H.
uvarum and T. quercuum. Furthermore, H. vineae was able to consume more sugar than P. kluyveri. In
general, the fermentations involving H. uvarum displayed high pH values, whereas those involving
P. kluyveri and the mixed P. kluyveri and H. uvarum resulted in high levels of residual sugar,
sugar/acid ratio, and glucose‐fructose consumption ratio. The pair P. kluyveri and H. uvarum
produced the highest concentration of glycerol. Noticeable variations in organic acids and
polyphenols were observed between the different fermentations. The analysis showed that acetate
esters contributed the most positively to the roasted and cooked aroma note in all ciders. This was
the first study evaluating the simultaneous fermentation of two non‐Saccharomyces yeasts to produce
cider. A recent study described the antagonistic and fermentative properties of Starmerella bacillaris.
The yeast proved to positively modulate cider volatile profile in the microfermentation trials [101].
Brettanomyces, Kluyveromyces, Schizosaccharomyces, Torulaspora, Zygosaccharomyces, and
Saccharomycodes have a negative influence on the product [102]. Brettanomyces may produce
2‐ethyltetrahydropyridine, 2‐acetyltetrahydopyridine, and 2‐acetylpyrroline, causing taste defects
and unpleasant smell in beverages.
Non‐Saccharomyces yeasts have high enzyme activity such as β‐glucosidase, esterase, and
β‐lyase. This enzyme activity contributes to a higher concentration of terpenes and thiols that may
add a positive fruity aroma and fragrance to the fermented product [103–106]. De Arruda Moura
Pietrowski et al. [107] and Wosiacki et al. [108] noted that the strains of Hanseniaspora sp. have a
positive impact on the aromatic profile of cider, thereby accentuating the beneficial role of these
yeasts. Nowadays, modern oenology is searching for novel strategies to reduce the final ethanol
content in fermented beverages. This trend is due to consumer demand for products with lower
ethanol content. The use of non‐Saccharomyces species reduces the initial ethanol content by
approximately 1–2% (v/v), depending on the yeast species and fermentation conditions [109–111]. In
addition, these yeasts can be used to regulate the acidity of drinks [112,113] as Saccharomyces yeasts
have no significant influence on acidity [114,115], and conventional chemical methods consist of the
addition of expensive and qualified products being of food quality.

5. Conclusions
This review emphasized the apple physicochemical and microbial composition and showed
how the fermentation can be affected by the first material composition. The present review also leads
the way for the optimization of the apple fruit fermentation by controlling the composition of the
raw material. In addition, the review underlined the importance of the microbial ecosystem of musts
and exposed how mastering the quality and the safety of cider production are reliant on a better
understanding of the mechanisms and yeast metabolism.

Author Contributions: All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Research council of Saint‐Joseph University of Beirut, grant number
FS103 and by the Lebanese National Council for Scientific research, grant number CNRS‐FS129.

Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 3698 11 of 16

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