International Journal of Food Properties

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Role of lactic acid bacteria on the yogurt flavour:

A review

Chen Chen, Shanshan Zhao, Guangfei Hao, Haiyan Yu, Huaixiang Tian &
Guozhong Zhao

To cite this article: Chen Chen, Shanshan Zhao, Guangfei Hao, Haiyan Yu, Huaixiang Tian &
Guozhong Zhao (2017) Role of lactic acid bacteria on the yogurt flavour: A review, International
Journal of Food Properties, 20:sup1, S316-S330, DOI: 10.1080/10942912.2017.1295988

To link to this article: https://doi.org/10.1080/10942912.2017.1295988

© 2017 Taylor & Francis Group, LLC

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INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. SI, S316-S330
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https://doi.org/10.1080/10942912.2017.1295988 Francis
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Role of lactic acid bacteria on the yogurt flavour: A review

Chen Chena, Shanshan Zhao c, Guangfei Haob'c, Haiyan Yua, Huaixiang Tiana, and
Guozhong Zhaob
a
School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, P.R. China; bState Key
Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu Province, P.R. China; cCollege of Agriculture, Hebei University of Engineering, Handan, P.R. China

ABSTRACT ARTICLE HISTORY

Considerable knowledge has been accumulated on the lactic acid bacteria (LAB) Received 31 August 2016
that affect the aroma and flavour of yogurt. This review focuses on the role of LAB Accepted 12 February 2017
in the production of flavour compounds during yogurt fermentation. The KEYWORDS
biochemical processes of flavour compound formation by LAB including Flavour; Glycolysis; Lactic
glycolysis, proteolysis, and lipolysis are summarised, with some key compounds acid bacteria; Lipolysis;
described in detail. The flavour-related activities of LAB mostly depend on the Metabolic engineering;
species used for yogurt fermentation, and some strategies have been developed to Proteolysis Yogurt
obtain more control of the flavourforming process. Metabolic engineering can be a
powerful tool to reroute the metabolic flux towards the efficient accumulation of
the desired flavour compounds with the knowledge of the complex network of
flavour-forming pathways and the availability of genetic tools. Further progress
made in the omics-based techniques and the use of systems biology approaches are
needed to fully understand, control, and steer flavour formation in yogurt
fermentation processes.

Introduction
Yogurt is one of the most popular fermented dairy products, and its consumption is increasing worldwide.[1]
According to the Euromonitor database, the yogurt production in 2015 reached 27.7 million metric tonnes, a
1.2-fold increase compared with the yield in 2010.[2] Its popularity is not only due to various health claims
and therapeutic effects but also for its sensory properties. The primary sensory attributes of yogurt include
texture, colour, and flavour.13,4 Yogurt is typically characterised as a smooth, viscous gel with a
characteristic taste of sharp acid and a green apple flavour. [5] Among these attributes, flavour has played an
important role in determining the acceptability and preference of yogurt for customers.
Flavour is the sensory impression of food or other substances and is determined primarily by the
chemical senses of taste and odour. Taste is the sensation produced when a substance in the mouth reacts
chemically with taste receptor cells located on taste buds in the oral cavity and can be expressed as sweet,
sour, salty, bitter, and umami.[6] Regarding odour, this is caused by one or more volatilised chemical
compounds, generally at a very low concentration, that humans or other animals perceive by the sense of
olfaction.[7] Taste and odour are complex phenomena in themselves, and their interaction with other sensory
properties increases the complexity of human perception. Furthermore, other factors, for example, textural
properties, sources of milk and fat content, also affect and interact with the flavour properties of yogurt,
which can be found in recent reviews.[8,9]

CONTACT Huaixiang Tian @ tianhx@sit.edu.cn School of Perfume and Aroma Technology, Shanghai Institute of Technology,
Shanghai 201418, P.R. China; Guozhong Zhao © zgzjiangnan@163.com @ State Key Laboratory of Food Science and Technology,
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, P.R. China.
© 2017 Taylor & Francis Group, LLC
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S317

For dairy products, the sensory properties depend largely on the relative balance of flavour compounds
derived from carbohydrate, protein or fat in the milk. The flavour components of yogurt include the volatile
and non-volatile compounds already present in the milk and specific compounds produced from milk
fermentation.[5,10] It has been suggested that more than 90 different volatile compounds have been identified
in yogurt, including carbohydrates, alcohols, aldehydes, ketones, acids, esters, lactones, sulphur-containing
compounds, pyrazines, and furan derivative. [10-12] In Table 1, a number of descriptions are given of some
flavour components and their thresholds in water (if known). However, not all of the volatile components
found in yoghurt are of sensory importance, as these components differ in concentration, only when its
concentration exceeds the threshold, the components can be perceived. Noticeably, the aroma threshold
values vary by several orders of magnitude (Table 1), which influences their perception by the human
senses. Although no final conclusion has yet been drawn, the major compounds commonly reported
responsible for imparting desirable flavour to yogurt are lactic acid, acetaldehyde, diacetyl, acetoin, and 2-
butanone.[5,8,13] Good flavoured yogurt results when proper levels of these compounds are produced. For
example, the optimal concentrations of acetaldehyde in yogurt ranged from 14 to 20 mg/kg, while less than
8.0 mg/kg resulted in weak flavour and too much acetaldehyde would lead to an “astringent” off-flavour
yogurt.[14] Furthermore, like many other dairy products, yogurt is prone to deterioration, especially under
improper storage conditions. Generation of volatile byproducts leads to off-flavours and makes the product
unsatisfactory for consumers.[15,16] Accordingly, a desirable flavour in yogurt means yogurt containing major
flavour compounds in proper levels without off-flavours.
A great majority of the flavour compounds produced in yogurt result from the activity of
microorganisms in starter cultures. The predominant organisms in these starter cultures are lactic acid
bacteria (LAB), for example, Lactococcus lactis, Lactobacillus species, Streptococcus thermophilus,
Bifidobacterium species, and Leuconostoc species.[17] During fermentation, these bacteria perform three
major biochemical conversions of milk components: (i) conversion of carbohydrate into lactic acid or other
metabolites (glycolysis), (ii) hydrolysis of caseins into peptides and free amino acids (proteolysis), and (iii)
breakdown of milk fat into free fatty acids (lipolysis). [18] These reactions also lead to the production of
various flavour and off-flavour compounds for yogurt catalysed by microbial enzymes.[19] In addition, a few
non-enzymatic reactions also occur, but they mainly derive from milk processing, for example, after
heating.[20]
Although the starter cultures could produce most key flavour compounds in yogurt, the amounts would
be far from sufficient during the fermentation process that adding food flavours is the conventional
approach to reach the demand of flavour acceptance by customers. However, there is a certain gap between
the external flavours and natural milk aroma.[14] In addition, food flavours do not meet the needs of
consumers for natural and environmental attributes of food products. In these days, the increase in intrinsic
potential to produce flavour compounds by LAB without adding food flavours (clean label) is the new trend
for yogurt-making. Other LAB are combined with yogurt starter cultures to produce yogurt with enhanced
nutritional properties and characteristic flavour. Metabolite formation and the effects of different LAB on
flavour compounds in yogurt have been investigated. [21,22] Furthermore, metabolic engineering has also been
applied in laboratory to change genetic and regulatory processes in the cells to increase their potential for
the formation of flavour compounds. [23,24] However, there is still a long distance between research and
application for this technology because of the legal restriction and the lack of consumer acceptance.
The aim of this article was to give an in-depth review of the role of LAB to yogurt flavour, including
flavour and off-flavour production from microorganism-mediated glycolysis, proteolysis and lipolysis, the
effect of different starter cultures on yogurt flavour, and the current development of metabolic engineering
for flavour enhancement. Finally, the recent literatures on methods to steer and control flavour formation by
LAB are also reviewed.
5318 C. CHENETAL.

Table 1. List of flavour compounds, description, and odour thresholds identified in yogurts.
Flavour compoundsa Descriptionb Odour threshold(mg/kg)c
Aldehydes
Acetaldehyde Ethereal, fresh, green, pungent 0.0079-0.039
Propanal Solvent, pungent 0.081-0.16
Butanal Pungent, green 0.007-0.017
3-Methylbutanal Cocoa, almond, green, malty, unripe, cocoa, malty 0.006
Hexanal Green, cut-grass 0.0091
Octanal Fat, soap, lemon, green 0.0009
Furfural Bread, almond, sweet 0.77
Benzaldehyde Almond, burnt sugar 1-4.6
Methional Cooked potato 0.0002
Pentanal Fruit, bread, sweet 0.022
2-Methylbutanal Fermented 0.003
Heptanal Green, sweet 0.06-0.55
Nonanal Sweet, floral, citrus, grass-like 0.04
(E)-2-Nonenal Green, fatty 0.0004
Decanal Fatty 0.03
Undecanal Fatty 0.01
Phenylacetaldehyde Floral sweet 0.004
Ketones
Diacetyl Buttery, creamy, vanilla 0.0011
Acetone Sweet, fruity 0.832
Acetoin Buttery 0.014
2-Propanone Sweet, fruity 0.832
2-Butanone Varnish-like, sweet, fruity 17-32
2-Pentanone Sweet, fruity, cheesy 1.38
2,3-Pentanedione Butter, vanilla, mild 0.02
2-Hexanone Floral, fruity 0.56
2-Undecanone Floral, rose-like, herbaceous 0.041 -0.082
1-Nonen-3-one Sweet,waxy 0.000000008
3-Penten-2-one Sweet, fruity 1.2
2-Heptanone Fruity, spicy 0.001 -0.01
3-Octanone Mushroom, fruity 0.028
Nonanone Grassy-herbal, green-fruity, floral 0.041 -0.082
2-Dodecanone Sweet, waxy 0.042-0.083
3-Hexanone Sweet, fruity 0.041 -0.081
1-Octen-3-one Mushroom-like, earthy, fruity 0.0005
Acids
Octanoic acid Wax, soap, goat, musty, rancid, fruity 0.91 -1.9
Propionic acid Vinegar, pungent, sour milk 2
Acetic acid Vinegar, pungent, acidic 25.59-26
Pentanoic acid Sweat 0.28
Hexanoic acid Pungent, rancid, flowery 1.8-1.84
Heptanoic acid 0.64-0.91
Decanoic acid Rancid 0.13
Butanoic acid Sharp, cheesy, rancid, sweaty, sour, putrid 1.40
Butyric acid Rancid, cheese, sweat 1.40
3-Methylbutanoic acid Rancid cheese, sweaty, putrid 0.1
Nonanoic acid Green, fat 4.6-9.0
Formic acid Pungent 1240-2440
Isovaleric acid Rotten fruit, mild, sweaty, rancid, faecal, putrid, flowery 0.1
Isobutyric acid Sweet, mild, rotten apple 0.05
Alcohols
1-Butanol Fruity, alcoholic 4.33
2,3-Butanediol Buttery 20-50
Methanol Alcoholic 800-1200
1-Propanol Alcoholic, pungent 5.7-7.8
2-Propanol Alcoholic 40-78
Ethanol Mild, ether 200
2-Heptanol Earthy, oily 0.041 -0.081
2-Butanol Wine 3.3
2-Furanmethanol Burnt, sweet, caramellic, brown 1.9-2.0
1-Penten-3-ol Fish, pungent 0.4
1-Pentanol Alcoholic, iodoform-like 0.12
(Continued)
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S319

Table 1. (Continued).

Flavour compoundsa Descriptionb Odour threshold(mg/kg)c

3-Methyl-2-butenol Sweet, fruity 0.25
3-Methylbutanol Fusel, alcoholic, pungent, ethereal 3.06
1-Octen-3-ol Mushroom-like 0.01
Guaiacol Bacon, phenolic, smoked, spicy
Esters 0.02
Ethyl acetate Solvent-like, fruity, pineapple 3.28-3.3
Methyl acetate Ethereal and solvent-like, estery, fruity, winey 1.5-47
Butyl acetate Pineapple 0.01 -0.1
5-Dodecalactone Fruit, sweet 0.06
Y—Dodecalactone Sweet, fruit, flower 0.007
Ethyl hexanoate Apple peel, fruit 0.001
Ethyl butanoate Fruity, sweet, banana 0.00018
Ethyl octanoate Fruity, banana, apple 0.015
Aromatic hydrocarbons
Benzene Aromatic 0.072
Toluene Paint 0.024
Ethylbenzene Phenol, spice 0.0024
1,3-Dimethylbenzene Plastic 1.1
1,4-Dimethylbenzene Leathery 1
Trimethylbenzene Leathery 0.5
Hydrocarbon
Dichloromethane 5.6
Limonene Lemon, orange 0.2
Ethenylbenzene Balsamic, gasoline 0.0036
Heterocyclic
2-Methylthiophene Sulphur 3
Pyrazine Roasted 300
Methylpyrazine Roasted 30
1 H-pyrrole Sweet, warm, nutty, ethereal
Furans 20
Furan Sweet 4.5-6
2-Methylfuran Ethereal, acetone, chocolate 3.5-4
2-Pentylfuran Green bean, butter
Sulphur compounds 0.006
Dimethyl sulphide Intense, lactone-like, sulphurous, cabbage 0.01
Dimethyl disulphide Boiled cabbage, cauliflower, garlic 0.0003
Dimethyl trisulphide Sulphurous, faecal 0.01
aFlavour compounds are from the following literature: Cheng, 151 Routray & Mishra,181 Beshkova et al.1581 and Thierry et al.1201 bOdour

descriptions are mainly sourced from the following website: http://flavornet.org/flavornet.html and http://www.thegoods
centscompany.com/
thresholds are mainly obtained from the literature which applied water as the matrix.1911

Biochemical routes of flavour compounds formation in yogurt

Flavour compounds produced from carbohydrate fermentation by LAB
In milk, lactose is present in substantial amounts in nature, and it is also the major energy and carbon source
for the growth of LAB. LAB convert lactose into lactic acid, which gives yogurt the characteristic acidic
taste. Variations in the metabolic products of lactose metabolism have yielded two main categories of
fermentation, homofermentation, and heterofermentation, depending on the LAB species, the substrate, and
the environmental conditions.[25] Homofermentative pathways generate lactic acid as the main end-product,
whereas heterofermentative metabolism results in some other metabolites, such as ethanol, carbon dioxide,
or acetic acid.[26] In addition, under certain conditions (carbon limitation, carbon excess of slowly
metabolised sugars, aerobic conditions), a homofermentative metabolism can be shifted to a mixed-acid
metabolism with various metabolites.[27-29] These metabolic products include several aroma compounds or
aroma precursors, such as acetaldehyde, ethanol, and diacetyl.
The typical aroma of yogurt is characterised chiefly by acetaldehyde, which exhibits a green apple or
nutty flavour. Its concentration ranges from 2.0 to 41 mg/kg, depending on the strains and process factors
used for yogurt fermentation. It has been reported that good flavour resulted only when greater than 8.0
mg/kg of acetaldehyde was produced in yogurt.[23,30] Some possible pathways of acetaldehyde synthesis
have been proposed (Fig. 1).[5,13] An important metabolic precursor is pyruvate, which can be catalysed by
S320 C. CHEN ET AL.

a-carboxylase or aldehyde dehydrogenase to generate acetaldehyde.[13] However, some researchers have

found that acetaldehyde could not be formed from pyruvate in yogurt, as no activity of these enzymes was
detected in S. thermophilus and Lb. bulgaricus.[31]
In fermented dairy products, various compounds with four carbon atoms are responsible for the typical
aroma of yogurt with its butter-like flavour. Collectively known as C4 compounds, they include diacetyl,
acetoin, and 2,3-butanediol.[32] They can be generated from glycolysis or citrate metabolism of several LAB
(Fig. 1), for example, Lactococcus, Leuconostoc, and Weissella species. Further details on the biochemical
processes in the production of C4 compounds in yogurt can be found in recent reviews. [3,5,8,17]
Among these C4 compounds, diacetyl is the most important flavour compound due to its low threshold.
The important effect of diacetyl on the aroma of milk has been recognised since 1929, when it was shown
that the distinctive aroma of fermented milk could be sensed just as the concentration of diacetyl reached 1
mg/kg.[33] The typical concentrations of diacetyl in yogurt range from 0.2 mg/kg to 3 mg/kg. [5,34] Both S.
thermophilus and Lb. bulgaricus are able to produce diacetyl, and strains of Lc. lactis subsp. lactis biovar.
diacetylactis can accumulate significant amounts of diacetyl due to their high capacity to metabolise
citrate.[35] Acetoin is the reduced form of diacetyl, and its flavour is considerably weaker than that of
diacetyl. However, acetoin is significant for

Figure 1. General metabolic pathways for the formation of the main flavour compounds of carbohydrate fermentation
by LAB. The pathways are generated according to the following literature: Cheng, [5] Thierry et al.[20] and Tamime &
Robinson.[13] The main flavour compounds are in bold. Key enzymes: CL, citrate lyase; OAD, oxaloacetate
decarboxylase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; ALS, a-acetolactate synthase; PFL,
pyruvate formate lyase; PDH, pyruvate dehydrogenase; ACDH, acetaldehyde dehydrogenase; ADHE, alcohol
dehydrogenase; ACK, acetate kinase; ALD, a-acetolactate decarboxylase; DR, diacetyl reductase; AR, acetoin
reductase; Tppi, thiamine pyrophosphate.
reducing the harshness of diacetyl and contributes to the mild creamy flavour. 151 2,3-Butanediol is the
reduced form of acetoin, and it makes limited contribution to the creamy or buttery attribute. 1361

Flavour compounds from amino acid conversion by LAB

The proteolysis of caseins is also an important biochemical pathway for flavour formation in yogurt. The
conversion comprises two steps: proteolysis and amino acid degradation. Proteolysis of casein by LAB is
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S321

initiated by cell-envelope proteinases (CEPs), which degrade the protein into oligopeptides (Fig. 2). For
some LAB, CEP activation requires the help of an extra-cytoplasmic chaperon called PrtM, which is a
lipoprotein essential to the autocatalytic maturation of CEPs. [37] The second stage of protein degradation is
the transport of di-, tri-, and oligopeptides into the cell (Fig. 2). Once casein-derived peptides are taken up
by LAB cells, they are further hydrolysed to amino acids by the actions of peptidases. Multiple copies of
peptidases can be encoded in one bacterial genome: for example, Lb. plantarum WCFS1 has 19 genes
encoding intracellular peptidases of different specificity,1381 while the genome of Lb. helveticus DPC 4571
has been found to include 24 genes encoding peptidases. 1391
Free amino acids produced by proteolysis may be converted to various flavour compounds including
ammonia, amines, aldehydes, phenols, indole, and alcohols, all of which may contribute

Figure 2. General conversion pathways of proteins relevant for flavour formation by LAB. The pathways are generated
according to the following literature: Smit et al.,[17] Sadat-Mekmene et al.[48] and Aleksandrzak-Piekarczyk et al.[25].Key
enzymes: CEP, cell envelope proteinase; PrtM, proteinase maturation protein; Opp, oligopeptide transporter; DtpT, ion-
linked transporter for di-and tripeptides; Dpp, ATP-driven transporter for di-and tripeptides; Pep, peptidase; AT,
aminotransferase; HycDH, hydroxyacid dehydrogenase; KaDH, a-keto acid dehydrogenase complex; PTA,
phosphotransacylase; ACK, acyl kinase; KdcA, a-keto acid decarboxylase; AlcDH, alcohol dehydrogenase; AldDH,
aldehyde dehydrogenase; EstA, esterase A.
S322 C. CHEN ET AL.

to yogurt flavour (Fig. 2). In particular, branched-chain amino acids (e.g. Val, Leu, Ile), aromatic amino
acids (e.g. Phe, Tyr, Trp), and sulphuric amino acids (e.g. Cys, Met) are the main sources of flavour
compounds derived from milk protein.[40] The first step of amino acid catabolism is transamination to their
corresponding a-keto acids. The a-keto acids can further undergo different enzymatic reactions: either a
reduction generating flavourless a-hydroxy acids, or a decarboxylation forming aldehydes, which can be
further reduced to alcohols, or undergo oxidative decarboxylation forming acyl-CoA and then carboxylic
acids.[25] Esters or thio-esters are then formed in reactions between alcohols and carboxylic acids, catalysed
by esterases or acyltransferases.[17] Another important conversion route of amino acids is initiated by lyases,
such as cystathionine ^- and y-lyases. These lyases are able to convert methionine to methanethiol, which in
turn can be converted into dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide via oxidation or
thioesters via esterase-catalysed reactions.[41] Although these sulphur compounds have significant impacts
on cheese sensory profiles, some of them are identified as off-flavours for yogurt products.[20] Threonine
aldolase belongs to another class of lyases and is able to convert threonine directly to acetaldehyde. [42] This
lyase pathway is thought to contribute greatly to the acetaldehyde pool of yogurt. [43] Furthermore, chemical
conversions also play an important role in flavour formation: for example, phenylpyruvic acid (the a-keto
acid of Phe) can be chemically converted to benzaldehyde. [44]

Flavour compounds from lipids in LAB

Lipids are also a source of aroma compounds for yogurt. Lipid breakdown is the main source for the
accumulation of free fatty acids (FAs), because most of the free FAs are derived from the decomposition of
triglycerides. Considerable quantities of short-chain FAs that are precursors of other aroma compounds are
produced from saturated FAs (Fig. 3).[45] Unsaturated FAs are oxidised in the presence of free radicals to
form hydroperoxides, which rapidly decompose to form hexanal or unsaturated aldehydes (Fig. 3).[5]
Unsaturated FAs also lead to the formation of 4- or 5-hydro- xyacids, which readily cyclise to cyclic
compounds.[46] The principal cyclic compounds in yogurt are y- and 6-lactones, which have 5- and 6-sided
rings, respectively, and are stable and strongly fruity

Triglyceride

Fatty acids

Saturated t atty acids unsaturated fatty acids

Thtohydrolasc
/> -oxidation
P -oxidatiot Oxidation
p-Keto acid Free fatty acids Ring closure Hy d roperoxid elase
Keto
’toacvldecarbox) 7acyl CoA
P -Ketoac
/- or o- lactones Aldehydes
Methyl ketone
Oxidation. Reduction
Reductase
4- or 5-Hydroxyacids Hydroperoxides
Secondary alcohol Acids Alcohols

Figure 3. General degradation pathways of lipids during milk fermentation by LAB. The pathways are generated according to the
following literature: Cheng[5] and Mcsweeney et al.[45].
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S323

flavoured. However, excessive or unbalanced lipid oxidation and lipolysis can also lead to off-flavour
especially during storage. Some products of lipid oxidation (e.g. aldehydes and ketones) give dairy products
the stale and “oxidised” flavours.
LABs are a source of esterase or lipases.[47,48] However, most LAB possess only intracellular esterases,
except Lb. fermentum and Micrococcus spp., in which extracellular esterases have been isolated. [49,50] As a
result, most esterases from LAB are unable to hydrolyse food lipids until their release from lysed cells.
Consequentially, the contribution of lipolysis to the flavour of yogurt is limited, compared with its
contribution to long-ripened cheeses.
Esterases of LAB can also catalyse the direct synthesis of flavour-active esters from glycerides and
alcohols via a transferase reaction (alcoholysis). [51,52] Research has shown that LAB can esterify ethanol
with butyric acid and hexanoic acid to form ethyl butanoate and ethyl hexanoate. [53] Interestingly, similar to
what is found for hydrolysis, di- and monoglycerides are preferred substrates, compared with triglyceride,
for the alcoholysis of esterases.[54]

Effects of different LAB on yogurt flavour

As yogurt-making is a complex process of milk transformation, the flavour of yogurt is influenced by
several factors, such as the starter cultures, processing parameters, source of milk and chemicals, and
additives used.[8,55] Among these factors, the starter cultures used for fermentation make key contributions
to the formation of the flavour compounds. Generally, the traditional yogurt culture is composed of S.
thermophilus and Lb. delbrueckii subsp. bulgaricus, and in several countries, the name “yogurt” is only
allowed for those products that are produced with starters containing strains of both species. [56] Although
the two microorganisms are able to grow individually in milk, they have a symbiotic interaction called
“proto-cooperation” in mixed cultures, which means that they are mutually beneficial during
fermentation.[56,57] It has been suggested that the level of flavour compounds is much greater in mixed
cultures than either of the two single cultures due to their associative growth and mutual stimulation. [13]
Beshkova et al. measured the production of flavour compounds when mixed cultures were used during
lactic acid fermentation and found that the maximum concentration of flavour compounds was reached
within 22 to 31 h of cooling stage. In the samples with 22 h cooling time, acetaldehyde predominated
(1415.0-1734.2 pg per 100 g), followed by diacetyl (165.0-202.0 pg per 100 g), acetoin (170.0-221.0 pg per
100 g), acetone (66.0-75.5 pg per 100 g), ethanol (58.0 pg per 100 g), and 2-butanone (3.6-3.8 pg per 100
g).[58] Some researchers have shown that in the mixed starter cultures, Lb. bulgaricus is mainly responsible
for acetaldehyde production, but others hold the opposite view. [10] In fact, both strains are able to produce
acetaldehyde, and the ability is strain specific.[59] Benozzi et al.[60] monitored the lactic fermentation driven
by different yogurt commercial starters using proton transfer reaction mass spectrometry. It was found that
nine volatile compounds showed considerable differences (statistically significant) among the four starters,
due to the starters’ different biosynthetic capacities for the formation of volatiles. De Bok et al. [21] also
found that the levels of the methylated sulphides and dimethyl trisulphide were relatively low in the
monocultures of Lb. bulgaricus and S. thermophilus, which suggests that the increased levels in the mixed
culture are the result of interaction between the two species. In addition, proteolytic activity plays an
important role in the proto-cooperation of the mixed cultures. However, Settachaimongkon et al. [61] showed
that only the non-proteolytic S. thermophilus strain performed proto-cooperation with Lb. bulgaricus. This
combination produced more aroma volatiles and non-volatile metabolites than pure cultures or cultures with
proteolytic S. thermophilus. From these results, it has been found that the association of these two
microorganisms affects the production of volatile and non-volatile molecules involved in flavour
development.
To increase the flavour of yogurt, strains belonging to the genera Leuconostoc and Lactococcus are
often incorporated as adjunct cultures. These strains can metabolise citrate to produce C4 compounds.[35]
The major compounds related to the utilisation of Leuconostoc are diacetyl, acetic acid, and ethanol, while
the flavour metabolites of Lc. lactis are diacetyl and acetoin. The most used strains are Lc. diacetylactis and
Leuconostoc citrovorum due to their high capacity to metabolise citrate.1621 Incorporation of these strains
into yogurt could enhance the butter-like flavour and improve the yogurt flavour quality.
Incorporation of probiotics into yogurt is a recent trend in functional dairy production. Most commercial
probiotics used in yogurt are strains belonging to the genera Lactobacillus and Bifidobacterium. In addition
S324 C. CHEN ET AL.

to their proposed health benefits, they also influence the flavour of yogurt into which they are incorporated.
As a well-known probiotic strain, Lb. rhamnosus GG has been added to yogurt in many countries. Although
it does not significantly influence the major aroma-forming volatile metabolites, it contributes to the yield
of volatile organic acids and alcohols during fermentation and increases the formation of non-volatile
organic acids and free amino acids during refrigerated storage. 1631 Some studies have compared the
properties of commercial fermented dairy products, such as Yakult@ and Actimel™, when fermented either
by Lb. casei alone or with the incorporation of yogurt cultures. For samples fermented only with Lb. casei,
the predominant compounds in the volatiles were acetic acid, acetoin, butyric acid, caproic acid, 2-
pentanone, and 2-butanone, while the volatile compounds typical of yogurt were absent. 1641 For samples
fermented with Lb. casei and yogurt cultures, the levels of 3-hydroxy-2- butanone and hexanoic acid greatly
increased.1651 As for other LAB, Lb. acidophilus has the ability to produce more acetic acid and
acetaldehyde166,671 and Lb. pentosus has been shown to efficiently produce ethanol, 2,3-butanedione and
acetic acid.1221
Bifidobacteria have been widely incorporated into dairy products in the last decade due to their potential
health benefits. Bifidobacteria use a distinctive “bifid shunt” for carbohydrate fermentation and convert
lactose into acetic acid and lactic acid in the proportion of 3:2. 1681 Thus, the final products obtained with
bifidobacteria often exhibit a characteristic aroma and a slightly acidic flavour.169,701 High levels of acetic
acid impart a “vinegary” taste that may not be accepted by consumers. 1131 However, the incubation
temperature greatly influences the ratio of acetic to lactic acid. In products fermented at 30 or 37°C, the
ratio between lactate and acetate was 3:2, but when strains were incubated at 42°C or 45°C, the ratio was
lower than the theoretical one.163,711 Furthermore, bifidobacteria have also been reported to contribute to
acetaldehyde and acetoin formation in yogurt.1721
These results suggest that in co-fermentation with traditional starters, the probiotic strains do not
influence the key aroma-forming volatile metabolites in yogurt, but produce different amount of metabolic
products after a defined fermentation time and temperature. A number of environmental factors including
composition of the culture medium, nutrient competition, and the interaction between microorganisms
affect the flavour formation in yogurt. It is essential to control the use level of probiotics and the
fermentation parametersto optimise the organoleptic effect of probiotic yogurt.

Metabolic engineering: Application for flavour enhancement

With the advent of molecular biology, various strategies have been applied to LAB to genetically engineer
strains that can over-produce certain flavour metabolites, such as acetaldehyde, diacetyl, and esters. As
stated before, there are many pathways for the production of acetaldehyde by yogurt bacteria, among which
acetaldehyde production from threonine catalysed by threonine aldolase is thought to be the key pathway.
In S. thermophilus, its serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, also possesses
threonine aldolase activity. Chaves et al. found that inactivation of the glyA gene resulted in a complete loss
of acetaldehyde formation, and overexpression of the gene in S. thermophilus results in an increase in
acetaldehyde production by 80-90%.123,731 Another attempt to improve acetaldehyde production through
metabolic engineering for Lc. lactis was reported by Bongers et al.1741 In that work, overexpression of
pyruvate decarboxylase (pdc) from Zymomonas mobilis in Lc. lactis, controlled by the NICE system,
rerouted pyruvate metabolism towards acetaldehyde. To further increase pyruvate availability, the NADH
oxidase gene (nox) was also overexpressed. The double mutant converted almost 50% of the consumed
glucose to acetaldehyde under anaerobic conditions.
Due to its importance in yogurt flavour, efficient diacetyl production has been the aim of several
metabolic engineering strategies for LAB. The key enzymes involved in diacetyl formation include a-
acetolactate synthase (encoded by the als or ilvBN genes), lactate dehydrogenase (encoded by the ldh gene),
and a-acetolactate decarboxylase (encoded by the aldB gene). Therefore, several approaches to improve
diacetyl production in Lc. lactis have been developed, such as the overexpression of the als genes or ilvBN
gene[75] and the inactivation of the ldh[62] or aldB[76,77] genes to increase the metabolic flux towards diacetyl.
However, the effects are limited, as the precursors of diacetyl, such as a-acetolactate and pyruvate are at the
centre of the metabolic pathways of organisms, and the change of a single gene may not be enough to
greatly direct the metabolic flux towards diacetyl production.
Consequently, various combined strategies have been developed. In a study with Lc. lactis, ldh
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S325

inactivation was combined with overexpression of the als gene in an attempt to produce high yields of
diacetyl. However, the result of the engineering was the production of very high amounts of acetoin rather
than diacetyl.[78] Thus, the activity of the ALDB enzyme, which catalyses the conversion of a-acetolactate to
acetoin, should be prevented. As attempts to combine both LDH and ALDB inactivation had found no
success, Monnet et al.[79] selected mutants using random mutagenesis that were deficient in a-acetolactate
decarboxylase and had low lactate dehydrogenase activity. The mutants were able to overproduce diacetyl,
with the maximum amount being 0.6 mmol/L. In addition, a combination of aldB deletion and increased
expression of ilvBN in a Lc. diacetylactis strain was found to increase the level of diacetyl to 0.53
mmol/L.[80]
Furthermore, the glycolytic flux in LAB is affected by the redox balance between NAD + and NADH.
NADH oxidase specifically utilises NADH and provides an extra route for the regeneration of NAD + under
aerobic conditions, thus leading pyruvate to be rerouted towards NADH-indepen- dent pathways, such as
the formation of a-acetolactate and diacetyl.[81,82] In 1998, Lopez de Felipe et al.[27] demonstrated that
overproduction of NADH oxidase can change Lc. lactis from a homo- lactic bacterium to a highly diacetyl-
producing bacterium. Hugenholtz et al.[83] described the combination of NOX overproduction with ALDB
inactivation in Lc. Lactis. Under aerobic conditions, 80% of the carbon flux was found to be rerouted via a-
acetolactate to the production of diacetyl, and the yield reached 1.6 mmol/L. To avoid the disadvantages
brought by the massive expression of controlling enzyme and elimination of the branching flux, Guo et al.
reported a novel strategy for fine tuning of lactate and diacetyl production in Lc. lactis through promoter
engineering.[24] Using selected promoters for the constitutive expression of the nox gene, NOX activity
increased by 58.17-fold compared with the wild-type strain under aerobic conditions. Meanwhile, the
corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM, which is the highest
value of diacetyl formation obtained by genetically modified LAB.
In metabolic engineering, little attention has been paid to dairy microorganisms for the production of
esters, although this approach has great potential for controlling ester biosynthesis in fermented dairy
products. An example of this is the cloning and overexpression of the esterase gene in Lc. lactis, with a
nisin-controlled expression system.[84] The enzyme can catalyse ester synthesis via an alcoholysis reaction
in an aqueous environment. As a result, the recombinants produced 2-5-fold ester yields compared with the
non-induced cells.[85]

Flavour improvement strategies and future prospects

The contribution of LAB to the formation of yogurt flavour depends on their intrinsic potential to produce
aroma compounds and on the way that this potential is (or is not) revealed during the fermentation process.
Although much has been done for increasing the intrinsic potential for flavour enhancement of LAB, there
are still some conundrums hindering the possible application in yogurtmaking. Firstly, several reviews have
summarised the flavour-forming pathways of LAB,[5’13’17’25’48] however, many of the known flavour
compounds cannot be traced back to their metabolic precursors due to our limited knowledge of the
complex network of flavour-forming pathways. Such issue will benefit from improvements of functional
annotation of the key enzymes in the formation of flavour compounds. Furthermore, the technological
progress made in high-throughput analysis methods, such as genomics, proteomics, and
metabolomics,[59,86,87] and the use of genome-scale metabolic models[88] has been driving the development of
new approaches to understand, control and steer aroma formation in dairy fermentation processes.
Second, the flavour-related activities of LAB mostly depend on the species used for yogurt
fermentation; however, some specific activities are found only in a few species. As an example, only some
specific variants of Lc. lactis (e.g. Lc. diacetylactis) are capable of citrate utilisation and diacetyl
accumulation. As a consequence, even small differences in the initial composition of a starter culture can
have profound effects on the profile of aroma compounds produced during fermentation. [21] Strategies have
been developed to explore the diversity on both genomic and phenotypic levels. New insights into the
potential of LAB to produce flavour compounds are expected from the comparison of complete genome
sequences of members of the same bacterial group, genus, or species. [89] In addition, the actual composition
of mixed and complex starter cultures is highly dynamic during the process of dairy fermentation, and the
microbe-microbe interactions are thought to be crucial for obtaining the desired product characteristics. The
classic example is the proto-cooperation and population dynamic of S. thermophilus and Lb. bulgaricus
S326 C. CHEN ET AL.

during growth in the yogurt, as stated before. Metagenomics and metatranscriptomics are increasingly
applied in food microbiology, whereas application of metabolomics approaches to food products enlarge
our view of in situ microbial metabolism and the varieties of metabolites.[20] The ultimate aim is to obtain
more control of the flavour-forming process executed by the fermenting LAB. To achieve this target, some
strategies are suggested: i) screening new bacteria for the detection of the presence and activity of key
enzymes involved in aroma production using omics-based approaches;[86] ii) changing the ratio among
different strains in mixed starter cultures to achieve the relative abundance of specific aroma-forming
strains at different steps in the fermentation process; and [19] iii) varying physicochemical parameters to
influence the microbial physiology leading to modulation of aroma production. [60]
The limited knowledge of the complex network of flavour-forming pathways of LAB also hampers the
progress of genetically modified LAB as starter cultures for industrial production of flavour
compounds.[83,89] For instance, although several metabolic engineering strategies have been designed to
improve diacetyl production by LAB, the effects are limited, as diacetyl is an intermediate metabolite, and
its synthesis and decomposition are controlled by many pathways and influenced by redox balance and
chemical modifications.[90] A genome-scale metabolic model that includes carbon and nitrogen metabolism
and flavour-forming pathways is crucial for understanding and designing metabolic engineering
strategies.[88] The combination of these strategies and the availability of numerous genetic tools for these
microorganisms will help reroute the metabolic flux towards the efficient accumulation of the desired
flavour compounds.

Conclusion
In conclusion, yogurt flavour development is a complex and dynamic biochemical process influenced by the
LAB used as starter cultures. The flavour-related compounds in yogurt are primarily derived from
microorganism-mediated glycolysis, proteolysis, and lipolysis. For flavour enhancement to yogurt-making,
strains of other LAB are incorporated as adjunct cultures and metabolic engineering are applied in
laboratory for the production of flavour-related compounds. Although the factors that determine the
formation of these compounds through yogurt fermentation remain poorly understood, the use of systems
biology approaches will help reveal the complex physiology of LAB and steer flavour formation during
yogurt-making.
Funding
This work was sponsored by Shanghai Rising-Star Program (No. 17QB1404200), “Shu Guang” project (No. 16SG50) supported
by Shanghai Municipal Education Commission and Shanghai Education Development Foundation and the Ability Construction
Project of the Science and Technology Commission Foundation of Shanghai (No. 15590503500).

ORCID
Shanshan Zhao http://orcid.org/0000-0002-7992-4331

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