Enzyme and Microbial Technology 143 (2021) 109722

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Enzyme and Microbial Technology

journal homepage: www.elsevier.com/locate/enzmictec

A review on preparation and chemical analysis of postbiotics from lactic

acid bacteria
Mehran Moradi a, *, Rahim Molaei a, Jonas T. Guimarães b
a
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
b
Department of Food Technology, Faculty of Veterinary Medicine, Federal Fluminense University (UFF), Niterói, Rio de Janeiro, Brazil

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

Keywords: Postbiotics may be defined as soluble metabolites released by food-grade microorganisms during the growth and
Cell free supernatant fermentation in complex microbiological culture, food or gut. It is rich in high and low molecular weight bio­
Lactobacillus logically active metabolites. There are still gaps concerning these substances, mainly how to use them for food
Probiotics
applications. Although the most recent work on preparation and application of postbiotics from several pro­
Functional food
Bioactive compounds
biotics are very encouraging, the suitability of postbiotics to combat microorganisms that deal with food safety
should be tested mainly by analyzing the chemical composition and conducting antagonistic tests. Consequently,
foods can effectively benefit from an identified postbiotic with a defined effect. This review approached the
recent advances in relation to the preparation of postbiotics from lactic acid bacteria. The function of different
instrumental analysis techniques and factors affecting the chemical composition of postbiotics were also
comprehensively reviewed.

1. Introduction gram-positive and catalase-negative microorganisms. The traditional

Currently, people have been associating healthy foods with a lower
quantity of additives and chemical substances, which are called “clean
label” products [1], and this awareness highlights the importance of
studying natural substances to ensure the food safety and fulfill the
consumers’ demand for healthier and natural foods. Antimicrobial
agents (AAs) are chemical or natural compounds that can increase safety
and extend the shelf life of food commodities by inhibiting the growth or
eliminating foodborne pathogens and spoilage microorganisms [2].
These agents comprise substances prepared mainly from four different
sources (Fig. 1). AAs of microbial origins in particular have gained
credibility over the years since it is a natural and renewable source.
Different microorganisms have been used as a source for the production
of AAs, among which probiotics are particularly noteworthy due to their
capacity to modulate the microbiota of humans and foods, besides its
recognized health benefits and lower risk of infections [3].
The probiotic microorganisms comprise different species, including
some lactic acid bacteria (LAB), Bifidobacterium spp., yeasts, and some
specific gram-negative strains [4]. LAB is a very heterogeneous group of
LAB are composed of different genera, predominantly Streptococcus,
Lactococcus, Lactobacillus, and Leuconostoc [5]. Lactobacillus spp. and
Bifidobacterium spp. are the most prominent probiotic bacteria (Fig. 2).
These generally recognized as safe (GRAS) agents are the dominant and
beneficial microorganisms of the colon and they are suitable for
amplification in the intestine, without provoking any significant side
effects. Other LAB and yeasts are also used as potential probiotic mi­
croorganisms (Fig. 2) [6], which can be found in functional products like
those manufactured with kefir cultures [7]. Actually, various strains of
Lactobacillus are used as primary and secondary (e.g., probiotic and
protective strains) starters in the production of fermented foods. Lb. casei
and Lb. acidophilus are probiotic strains used in some renowned pro­
biotics foods (e.g., BIO®, Actimel®, LC1®, and Yakult®). To stimulate
beneficial bacteria to grow in the gut and produce their bioactive sub­
stances, soluble edible oligosaccharide carbohydrates [e.g.,
Fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS)] and
other compounds, which are commonly known as prebiotics, may also
be added to the food [8].
LAB are capable of producing several substances, among which many

Abbreviations: AA, antimicrobial agents; LAB, lactic acid bacteria; GRAS, generally recognized as safe; Lac., Lactococcus; Lb., Lactobacillus; Leuc., Leuconostoc; L.,
Listeria; Ped., Pediococcus; Ps., Pseudomonas; Salm., Salmonella; Staph., Staphylococcus; Strep., Streptococcus; B., Bacillus; Cl., Clostridium; Ent., Enterococcus; E.,
Escherichia; Kl., Klebsiella; CFS, cell-free supernatant; BLIS, bacteriocin-like inhibitory substances; EPS, exopolysaccharides; MRS, De Man Rogosa and Sharpe.
* Corresponding author.
E-mail address: m.moradi@urmia.ac.ir (M. Moradi).

https://doi.org/10.1016/j.enzmictec.2020.109722
Received 17 August 2020; Received in revised form 25 November 2020; Accepted 28 November 2020
Available online 3 December 2020
0141-0229/© 2020 Elsevier Inc. All rights reserved.
M. Moradi et al.
Fig. 1. Classification of natural antimicrobial compounds originated from an­
imal, microbial and plant sources as well as algae and mushroom.
can inhibit the growth of other microorganisms (Table 1). The AAs of
LAB have significant importance from the point of view of food safety
and quality so that some of them are currently being used by the industry
such as the nisin, which is considered a bacteriocin [9]. Antimicrobial
substances from LAB can be broadly categorized by their molecular
weight into those that have low molecular weight (H2O2, organic acids,
acetoin, acetaldehyde, etc.) and those that have a high molecular weight
(e.g., bacteriocins) [10]. Bacteriocins, organic acids, enzymes, alcohols
and low molecular mass substances (e.g., reuterin derived from Lb.
reuteri) are the main metabolites responsible for the antimicrobial action
of LAB. Bacteriocins are the most studied postbiotic metabolites [11,12].
The extracellular polymers of microorganisms, known also as exopoly­
saccharides (EPS), have also gained more attention for food applica­
tions. Although EPS of LAB displays many techno functional
applications [13], some biological properties of this novel additive, as
antimicrobial and antioxidant activities, have also been investigated
[14].
Fig. 2. Some well-known probiotics microorganisms with perceived functional and nutritional benefits in food and human.
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Enzyme and Microbial Technology 143 (2021) 109722
The main challenge of using live starter/probiotic cultures directly
on the food is their incompatibility with different matrices and envi­
ronments that hinder their growth and survival in food. As an alterna­
tive, the use of postbiotics avoids the negative interactions between
primary and secondary starters and the food [15]. Therefore, using
postbiotics instead of the living microorganism is possible to take full
advantage of their broad-spectrum antimicrobial activity, the synergis­
tic activities between organic acids and other metabolites, and the high
heat stability of the postbiotics mixture [16].
For industrial applications, postbiotics are recognized as superior to
probiotics due to the defined chemical composition, safety, ease of use
and storage, stability in a broad range of pH and temperature and broad-
spectrum antimicrobial activity [17]. Currently, there are different
commercially available postbiotics with various applications other than
food (Table 2), however, little is known about their methods of prepa­
ration and analysis, and the factors affecting the production of each
postbiotic compound, which may be a barrier to the new researches and
the large-scale application in the food industry. Recently, the applica­
tion of postbiotics mixtures and individual postbiotics in food safety
[16], biotherapy and functional foods [18,19], nutrition [20,21], dis­
eases prevention [22,23], animal feed and pharmaceutical industries
[24], and allergy treatment [25] have been reviewed; however, there is
still a gap in the literature on the steps prior to the application of post­
biotics in food.
2. Definition of postbiotics
Different nomenclatures have been used to describe the fermentation
functional compounds produced by food-grade microorganisms [26,27],
however, postbiotics (metabiotics or biogenics) is the most used today.
There is still no consensus on the definition of postbiotics, but most
studies consider that they must provide any physiological benefits to the
host, which are sometimes similar to the effects provided by the living
probiotics [28,21,22]. However, due to the unclear definition and the
wide bioactivity of metabolites produced by probiotic and non-probiotic
bacteria, the term postbiotic can be defined as any soluble factor (pro­
ducts/metabolic by-products of microbial metabolisms or substances
produced by the action of LAB on culture/food ingredients; e.g.,
bioactive peptides) secreted by food-grade microorganisms or released
after cell lysis, during the growth and fermentation in complex
M. Moradi et al. Enzyme and Microbial Technology 143 (2021) 109722

Table 1
The main inhibitory substances produced by different lactic acid bacteria.
Compounds name LAB strains Biological Analytical method Reference
benefit

Organic Acids
Pyruvic acid Lb, Ped Antimicrobial GC-MS [51]
Lactic acid Lb, Ped Antifungal, G+− LC- SPD/ GC-MS [94,95]
Acetic acid Weissella, Lb Antifungal, G+− GC-MS/ LC- SPD [96]
Tartaric acid Lb Antifungal, G+− [94]
Phenylacetic acid Antifungal, G+− MS LCQ and NMR [97]
Benzeneacetic acid Lb Antifungal LC-MS/1H NMR and 13C [57]
NMR
Succinic acid Lb, Ped, Lact Antifungal, G− HPLC-DAD/ GC-MS [51,83]
Phenyllactic acid Weissella, Lb Antifungal, G+− GC-MS [51,68]
Hydrocinnamic acid Weissella, Lb Antifungal GC-MS/ LC-FTMS/ LC-MS/ [68,76,98]
MS
Caffeic acid Weissella, Lb Antifungal HPLC-UV/DAD [60]
4-Hydroxyphenyllactic acid Weissella, Lb, Antifungal LC-FTMS/ GC-MS [51,98]
Ped
Indole-3-lactic acid Weissella, Lb, Antifungal HPLC-UV/ GC-MS [51,99]
Ped
Citric acid Lb, Ped Antifungal, G+− LC- SPD/ GC-MS [51,94]
Malic acid Lb G+− LC- SPD/GC-MS [58,94]
p-Coumaric acid Lb Antifungal, G+− GC-MS/ LC-FTMS [68,100]
Caftaric acid Lb Antifungal LC-MS/MS [76]
Azelaic acid Weissella, Lb Antifungal, G+− GC-MS/ LC-FTMS [68,98]
Propionic acid Lb, Lac, Strep Antifungal, G+− GC-MS/ LC-MS/MS [76,101]
Phenylpyruvic acid Lb Antifungal LC-MS/MS [76]
Vanillic acid Lb, Lac, Strep Antifungal, G− LC-FTMS [98,100]
Formic acid Lb Antifungal, G+− LC-MS [57]
Fatty Acids
2-Hydroxyisocaproic acid Weissella, Lb Antifungal, G+− LC-FTMS/ GC-MS [51,98,
100]
Oleic acid Weissella, Lb, Antifungal, G +−
GC-MS [51,96]
Ped
Butyric acid Lb, Lac, Strep Antifungal, G+− HPLC-DAD [83]
Linoleic acid Lb, L Antifungal, G+− HPLC-MS [102]
6-Octadecenoic acid methyl ester Lb Antimicrobial GC-MS [50]
3-hydroxydecanoic acid Lb Antifungal GC-MS/ LC-FTMS [68,100]
Myristic acid (tertadecanoic acid) Lb Antifungal, G+ GC-MS [101]
Sebacic acid Lb Antifungal GC-MS [101]
3-hydroxydodecanoic acid Lb, Weisella Antifungal LC-FTMS/HPLC [98,103]
3-hydroxytetradecanoic acid (β-Hydroxymyristic acid) Lb Antifungal HPLC [103]
Capric acid Lb, Weisella Antifungal, G+ LC-FTMS [100]
Bacteriocins
Bacteriocin LF-BZ532 Lb G+− RP-HPLC [104]
Bacteriocin SLG10 Lb G+ RP-HPLC [105]
Bacteriocin BMP32r Lb G+ RP-HPLC [106]
Bacteriocin DY4− 2 Lb G− Gel chromatographic, [107]
HPLC
Salivaricin mmaye1 Lb G+− RP-HPLC [108]
Other metabolites
2,4-Di-tert-butylphenol, Heptacosane Weisella Antifungal, G+− GC-MS [96]
1
Phenylalanine Lb G− H-NMR [109]
2-Methyldecane, Pentadecane,2,4-bis(1,1-dimethylethyl)phenol, Dotriacontane Lb Antifungal, G+− GC-MS [50]
Sinapic acid, 2-Deoxycytidine, Cyclo (L-His-L Pro), Phosphoric acid, Cyclo (L-Tyr-L Pro),3,5- Lb Antifungal LC-MS/MS [76]
Di-O-caffeoylquinic acid
Salicylic acid (2-hydroxybenzoic acid) Lb Antifungal, G− LC-FTMS [98]
Diacetyl(2,3-butadione) Lb, Lac, Ped, Antifungal, G+− Headspace GC-MS [110]
Leuc
Acetaldehyde Lb, Lac, Ped, Antifungal, G+− Headspace GC-MS [110]
Leuc
Acetoin Lb, Lac, Ped, Antifungal, G +−
Headspace GC-MS [110]
Leuc
Hydrogen peroxide, carbon dioxide, ethanol LAB Antifungal, G+− HPLC [111]
pyrrolo [1,2-a] pyrazine-1,4-dione Lb Biofilm removal GC-MS [46]
Secretory proteins Weisella G+− MALDI-TOF MS [61]
(trans, trans)-3,4-dihydroxycyclohexane- 1-carboxylic aci Lb G+− NMR [68]
Gallic acid Lb G+ HPLC [112]

*G+− gram positive and negative. Lac: Lactococcus; Lb: Lactobacillus; Leuc: Leuconostoc; Ped: Pediococcus; Ent: Enterococcus. Strep: Streptococcus. B: Bifidobacterium.
GC–MS: Gas Chromatography–Mass Spectrometry; NMR: Nuclear Magnetic Resonance; MALDI-TOF MS: Matrix Assisted Laser Desorption Ionization-Time of Flight
Mass Spectrometry; HPLC: High Performance Liquid Chromatography; RP-HPLC: Reversed-phase HPLC; LC-FTMS: Liquid Chromatography Fourier Transform Mass
Spectrometry; LC–MS/MS: Liquid Chromatography with tandem mass spectrometry; DAD: Diode-Array Detector; ESI-MS: Electrospray Ionization mass Spectrometry.

3
M. Moradi et al. Enzyme and Microbial Technology 143 (2021) 109722

Table 2
Commercially available postbiotics.
Name Corresponding microorganism Active ingredient Application Manufacturer

Del-Immune Lb. rhamnosus V Cell wall and DNA fragments Immunomodulator Pure Research
V® Products LLC
Hylak® Ent. faecalis, Lb. acidophilus, Lb. helveticus and E. coli Bacteria-free liquid containing acetic Antimicrobial activity on pathogenic Ratiopharm/
forte acid, short-chain fatty acids, glutamic bacteria in gut Merckle GmbH
acid, formic acid, riboflavin and
glutamine
CytoFlora® Lb. casei, Lb. plantarum, Lb. acidophilus DDS-1, Lb. reuteri, Probiotics tincture containing cell Balances intestinal dysbiosis, creating BioRay Inc,
Lb. salivarius, Lb. rhamnosus, Lb. sporogenes, Lb. bulgaricus lysate of probiotic bacteria an environment for healthy intestinal
Lb. acidophilus, B. bifidum, B. infantis, B. bulgaricus, and flora to colonize
Strep. thermophiles
Aktoflor C Unknown Lysine, lactic acid, succinic acid Reducing intestinal disorder Solopharm
symptoms company
Zakofalk Unknown Inulin and butyrate Immunomodulating Dr. Falk Germany

Lac: Lactococcus; Lb: Lactobacillus; Leuc: Leuconostoc; Ped: Pediococcus; Ent: Enterococcus; Strep: Streptococcus. B: Bifidobacterium.

microbiological cultures (in this case called cell-free supernatant; CFS),
food or gut [20,26,29,24,30] (Fig. 3). From this perspective, postbiotics
prepared from LAB comprise hundreds of individual metabolites used
either as a crude concentrate (extract) or as a semi-purified form.
Postbiotics of probiotic cultures seem to be a rich source of bacte­
riocin and bacteriocin-like inhibitory substances (BLIS) with antago­
nistic activity on major foodborne pathogens. Moreover, in some
context, postbiotics were defined as cell metabolites and cell wall-
derived substances (exopolysaccharide; EPS, teichoic acids, peptido­
glycans, polar lipids, glycolipids, peptidoglycan, and proteins) [31] of
microorganisms of which the cell walls were ruptured following lysis by
heat treatment, enzymes application or ultrasonic processing [26,32].
Cell-derived ingredients generally display immunomodulation and
antiproliferative activity in the host [19]. The term paraprobiotics
(ghost probiotics) is used to refer to the non-viable or inactivated pro­
biotic strains, either in intact or ruptured forms [20,33]. Unfortunately,
there is no agreement on the definition of postbiotics, but it generally
defines as biological materials that contain probiotic-derived metabo­
lites or cell wall-derived materials (e.g., peptides, teichoic acids, surface
protruding molecules, exopolysaccharides, biosurfactants, and plas­
malogens) without the presence of viable microorganisms or cell
structures [16,18,19].
3. Preparation of postbiotics from LAB
To obtain a postbiotics mixture, in most cases, the researchers pre­
pared a solution called cell-free supernatant, with or without cell lysis,
containing 1) products from microbial metabolism, 2) metabolites syn­
thesized by the action of LAB on culture/food ingredients or 3) struc­
tural substances produced by LAB and used as postbiotics mixture [18,
19]. The quantity and type of products are mainly related to the type of
bacterial strain, type of culturing medium, bacterial treatment after
propagation, etc. In all published works on the application of postbiotics
in the food, no post-propagation treatments were applied. In this case, as
illustrated in Fig. 4, postbiotics contains only soluble factors such as
products or metabolic by-products that have been secreted into the
medium during the bacterial growth [30]. However, in some works,
after propagation, bacterial cells were subjected to lysis by enzymes,
thermal, sonication, and high-pressure treatments or a combination of
them (Fig. 4) [19,34,35]. These treatments introduce some additional
intracellular metabolites and cell wall- derived materials into the post­
biotics mixture and give new functionality to the obtained postbiotics. In
both treated and non-treated postbiotics mixtures, the obtained solution
is centrifuged or ultrafiltrated in order to separate bacterial cells from
postbiotics metabolites [24].
Regardless of the type of preparation (bacterial lysis or not), at the
laboratory and industrial level, there are several important points to be
considered for postbiotics production such as the fermentation medium,
bacterial propagation, harvesting and concentration methods, preser­
vation, and method of application. Generally, LAB, whether as the pri­
mary or secondary starter, are used for postbiotics preparation;
however, the tendency to non-starter LAB has greatly increased and
most attention needs to be paid to the safety of final products. Post­
biotics is most commonly prepared by culturing the LAB in culture

Fig. 3. Schematic illustration for postbiotics concept from lactic acid bacteria in microbiological culture media, food and gastrointestinal tract.

4
M. Moradi et al.
Fig. 4. The five steps of preparing postbiotics (cell free supernatant) mixture form lactic acid bacteria (LAB). 1: resuscitation of LAB, 2: propagation of LAB, 3:
treatment, 4: postbiotics harvesting and 5: postbiotics concentration. MRS stands for De Man, Rogosa and Sharpe.
media [mainly De Man, Rogosa and Sharpe (MRS) broth], followed by
an extraction step (centrifugation at 4000− 12000 g for 10 min at 4 ◦ C or
dialysis) (Fig. 4) (Table 3) [17,36,37]. Postbiotics, if prepared in MRS,
produce a bacteria-free and acid fermentate with brown to brownish
yellow color [38] (Fig. 5). Recently, the suitability of other alternatives
for the production of postbiotics at the industrial scale was also reported
(Table 3). In one study, different dairy-based ingredients including
low-heat milk and milk permeate have optimized based on incubation
time and temperature, as a fermentation medium for Lactobacillus spp. in
order to prepare antifungal postbiotics solutions (or fermentate) for food
application [39]. Due to differences in protein and fat content, post­
biotics mixtures prepared in low-heat milk revealed remarkable anti­
fungal activity. Postbiotics prepared in both substrates had suitable
compatibility with dairy food applications. Skim milk (10 %) has suc­
cessfully applied for the production of postbiotics of an adjuncts culture
(i.e., DSM-100 H) at an industrial scale [37]. The suitability of some
alternative dairy substrates such as whey and buttermilk as a propaga­
tion medium for different commercial probiotics were also investigated
[40].
However, postbiotics can be prepared in both liquid and dry (spray-
dried and lyophilized) forms [4]. Lyophilization process is the dominant
preservation method, although spray drying is also used [37]. However,
the method of application of postbiotics depends on the type of food.
Generally, pasty lyophilized postbiotics exhibit enhanced antimicrobial
activity and minimize the increase of moisture in food compared to the
postbiotics solution [41]. In addition, spraying of postbiotics on the
surface of food may serve as an efficient alternative in order to inhibit
the growth of spoilage and pathogen microorganisms [39]. For sterili­
zation of postbiotics, the solution is typically filtered with a 0.22 or
0.45-μm pore size filter and kept at -20 to − 80 ◦ C until its use. In the case
of lyophilized and spray dried concentrates, the solution is sterilized
before use. A sterile buffer solution (phosphate buffer saline; PBS,
0.01 M phosphate, pH 7.2) is used to resuspend pasty postbiotics [42].
3.1. Modification of postbiotics mixture for antimicrobial application
The antimicrobial performance of each AA in postbiotics can be
elucidated by treating the postbiotics solution. Initially, a filter-
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Enzyme and Microbial Technology 143 (2021) 109722
sterilized (0.22 μm) postbiotics solution should be divided into three
equal fractions. The antimicrobial function of organic acids can be
monitored by neutralizing the pH of the postbiotic solution (pH 6.5–7)
with NaOH 1 N [43], while the hydrogen peroxide-dependent activity
can be checked by the addition of catalase (5 mg/mL or 300 IU/mL) into
the second solution (or previously neutralized solution) for 1 h at 37 ◦ C
[43,44,45]. For bacteriocin and BLIS assay, the third solution (or pre­
viously NaOH-catalase- treated solution) should be treated with
1 mg/mL of a proteolytic enzyme (trypsin, α-chymotrypsin, pepsin,
papain or proteinase K) at 37 ◦ C for 2 h followed by incubation at 100 ◦ C
for 5 min [46].
4. Chemical analysis of postbiotics
From a fundamental point of view, the suitability of postbiotics to
fight harmful microorganisms that deal with food safety should be tested
primarily by analyzing their chemical composition and conducting in
vitro antagonistic tests. Metabolomics involve the identification of me­
tabolites in biological systems, then, it could help to investigate post­
biotic metabolites originated from LAB [47]. The quantity and quality of
postbiotics and molecules harboring antagonistic activities should be
tested using analytical procedures [29]. In this regard, foods can effec­
tively benefit from an identified postbiotic with defined effects. There is
much literature available addressing the existence of a wide range of
metabolites in postbiotics [20]. Accordingly, given the complexity of
biological compounds with different polymerization degrees and
glycosidic bonds, the qualitative and quantitative identification and
characterization of the postbiotic matrix generally requires sophisti­
cated equipment with several concentration/clean up steps.
For the qualitative and/or quantitative analysis of postbiotics, a large
number of instrumental techniques and innovative approaches have
been applied (Table 1). The appropriate method is usually selected
based on the analytical goals and the pursued type of characterization
[17]. Herein, we reviewed various analytical techniques used to
examine the chemical composition of postbiotics.
M. Moradi et al. Enzyme and Microbial Technology 143 (2021) 109722

Table 3
Postbiotic preparation and application from various LAB strains under different conditions and their potential application.
Name of LAB Postbiotic using Medium Culture conditions/postbiotics preparation Application Reference
form

Lb. acidophilus Solution MRS broth At 37 ◦ C for 24 h/ Centrifugation (6000 g for Antibacterial and antibiofilm [113]
15 min) potential
Lb. curvatus BCS35 Solution and MRS broth At 30 ◦ C for 16 h /Centrifugation Food ingredient in fresh fish [36]
lyophilized forms (12,000 rpm, 4 ◦ C, 10 min)
Lactobacillus spp. isolates- Solution in MRS broth At 37 ◦ C overnight /Centrifugation (10,000 g Antibacterial and antibiofilm [114]
Lb. acidophilus neutralized and for 15 min at 4 ◦ C) potential
heat-treated forms
Different LAB Solution MRS broth At 37 ◦ C for 6 h /Centrifugation (9000 rpm, Antibacterial and inhibition of [115]
4 ◦ C, 20 min) biogenic amine production in
vitro
Lb. acidophilus A9, 08 and Solution Buttermilk and whey based At 37 ◦ C for 10 h None [40]
H5, Lb. paracasei A13 media with or without 0.3 %
and LS and Lact. casei yeast extract, with 0.3 % yeast
extract plus 1% glucose
Lb. acidophilus LA5 Lb. casei Solution MRS broth At 37 ◦ C for 48 h in CO2 incubator/ Antimicrobial and biofilm [43]
431 Centrifugation (4000 g for 10 min) removal activity
A LAB with antifungal Lyophilized MRS broth At 37 ◦ C for 24 h /Centrifugation (8500 rpm, Control of aflatoxin producer [50]
activity 4 ◦ C, 20 min) Aspergillus
LAB Lyophilized Low-heat milk and milk Different formulations Antifungal activity on semi- [39]
permeate hard cheese molds
Lb. plantarum Solution in MRS broth At 37 ◦ C for 24 h /Centrifugation (8000 rpm, In vitro reduction of biogenic [116]
neutralized and 4 ◦ C, 10 min) amines
heat-treated forms
Lb. acidophilus LA-5® Solution MRS broth At 37 ◦ C for 48 h, anaerobic /Centrifugation Biosynthesis of carbon dots [117]
(4000 rpm, 4 ◦ C, 10 min)
Lb. rhamnosus Lyophilized MRS broth At 37 ◦ C for 18 h, anaerobic /Centrifugation Development antimicrobial [118]
(8000 rpm, 4 ◦ C, 10 min) packaging
Lb. brevis WK12 and Leuc. Solution MRS broth At 30 ◦ C, and aerobic anaerobic Washing sanitizer in leaf [119]
mesenteroides WK32 /Centrifugation (5000 rpm, 4 ◦ C, 15 min) vegetable
Selected Lactobacillus spp. Lyophilized 10 % Skim milk Laboratory scale production in bioreactor at Functional food ingredients [37]
43 ◦ C with an agitation rate of 200 rev/min
and CO2 sparring (02 L/min) for 48 h/
Centrifugation (4000 rpm, 8 ◦ C, 30 min)
Lb. plantarum Lyophilized MRS broth at 37 ◦ C for 24 h in CO2 incubator/ Development antimicrobial [72]
Centrifugation (4000 g for 10 min) packaging for ground meat
Lb. plantarum Cys5− 4 Solution MRS broth At 32 ◦ C for 30 h /Centrifugation (13,000 g Antibacterial activity in [120]
4 ◦ C for 20 min) artisanal beverages
Lb. actobacillus reuteri, Lb. Solution in MRS broth At 37 ◦ C until reaching stationary phase/ Down-regulate the gene [121]
plantarum, Lb. fermentum neutralized form Centrifugation (10,000 rpm, 4 ◦ C, 10 min) expression of some virulent
factors in multidrug-resistant
foodborne pathogens
Lb. acidophilus (EMCC Lyophilized MRS broth At 37 ◦ C for 48 h /Centrifugation Control of food borne pathogen [122]
1324), B. bifidum (EMCC (10,000 rpm, 10 min) on soft feta cheese
1334) and Lb. plantarum
(EMCC 1845)
Lb. salivarius Lb. acidophilus Lyophilized MRS broth At 37 for 48 h in CO2 incubator / Improving shelf life of ground [38]
Centrifugation(at 4000 g for 10 min) meat

Lb: Lactobacillus; MRS: De Man, Rogosa and Sharpe.

Fig. 5. The appearance of postbiotics mixture in solution (A) and lyophilized (B) forms prepared from Lb. salivarius in De Man, Rogosa and Sharpe (MRS) Broth.

6
M. Moradi et al.
4.1. Gas chromatography (GC)
GC is the most frequently used analytical tool for quantitative and
qualitative analysis of free fatty acids, volatile compounds (e.g., diace­
tyl, acetoin and dimethyl sulfone, 2-butanone), and organic acids in
postbiotics. Moradi et al. (2019) have utilized the GC–MS approach to
identify the chemical composition of postbiotics derived from Lb. aci­
dophilus LA5, Lb. casei 431 and Lb. salivarius. However, depending on the
sample type and detection technique, additional steps are usually
required before GC–MS analysis. For example, the volatile profile of
postbiotics from Lb. casei was fully characterized through the headspace-
solid phase microextraction (HS-SPME) GC–MS technique and the au­
thors confirmed the presence of 62 compounds, including alcohols,
terpenes, and norisoprenoids, acids, ketones and esters in the postbiotics
[48]. In another study, a GC equipped with a flame ionization detector
(GC-FID) was used to study the short-chain fatty acid (SCFA) content of
postbiotics of four bacterial strains, Lb. acidophilus, Lb. helveticus, E. coli,
and Ent. Faecalis [49]. In the standard process of the analysis of SCFA
with GC-FID, the derivation of fatty acids by boron trifluoride-ethanol
complex is necessary [49].
For the quantification of organic acid by GC–MS, the postbiotics
sample should primarily be esterified with absolute ethanol and sulfuric
acid 97 % v/v [50]. In another work, the organic acid profile was
determined in the postbiotics from Lb. pentosus K34 and Ped. lolli PL24
by GC–MS following methoxime/ tert-butyldimethylsilyl derivatives
[51]. Fatty acids and volatile compounds produced by Lb. rhamnosus
with a potential antifungal activity were evaluated using GC-FID and
GC–MS methods, respectively [52]. A review paper was published
describing the capability of GC–MS as an interesting method to inves­
tigate the monomeric composition of several EPS produced by different
LAB such as Lac. lactis spp. cremoris, Strep. thermophilus, Lb. delbrueckii
spp. bulgaricus, Lb. helveticus, Lb. sake, Lb. reuteri, Lb. rhamnosus, and
B. longum [53].
4.2. Liquid chromatography (LC)
The high-performance liquid chromatography (HPLC) method is also
one of the most utilized techniques for qualitative and quantitative
investigation of the chemical composition of postbiotics from LAB. The
widespread application of this assay is related to the high potency, pu­
rity, and performance features of HPLC. The technique is highly rec­
ommended when we need a concomitant analysis of different organic
acids (acetic, malic, lactic, acetic, and other acids) in the postbiotics. The
coupling of HPLC with a proper detector like ultraviolet (UV), ultra
violet-diode array detector (UV/DAD) refractive index (RI), mass spec­
troscopy (MS), and pulsed electrochemical detection (PED) is the key to
success in HPLC performance [54]. Patil et al. (2019) utilized HPLC-RI
to determine the presence of various mono- and disaccharides, EPS,
and vitamins in the postbiotics. As well, HPLC-UV/DAD technique was
successfully employed to examine the antifungal compounds present in
the growth media of Lb. amylovorus DSM19280 [55].
One of the promising approaches is the use of ultra-performance
liquid chromatography (UPLC), which represents superior separation
and identification capacity for postbiotics owing to the high efficiency,
resolution, sensitivity, and accuracy along with low use of solvents [29].
UPLC has been applied in the characterization of intracellular compo­
nents such as enzymes and proteins of Lb. plantarum [56]. In another
study, the qualitative analysis of the antifungal compounds (3-phenyl­
lactic acid, benzeneacetic acid, and 2-propenyl ester) present in the Lb.
plantarum IMAU10014 postbiotics was performed through UPLC system
equipped quadrupole ion trap mass spectrometer [57]. Furthermore,
Lee’s group employed UPLC-MS (UPLC-Q-TOF) to identify the cell
metabolite profiles of Lb. sakei and its culture media at different growth
phases [58]. An antifungal organic acid in the binary combinations of
Lactobacillus spp. (Lb. plantarum L244, Lb. harbinensis L172, Lb. rham­
nosus CIRMBIA1113, and Lb. brevis L128) using LC–Q–TOF were
7
Enzyme and Microbial Technology 143 (2021) 109722
quantified [59]. In another study, the analysis of the metabolites in LAB
cultures using a liquid chromatography linear ion trap quadrupole
Orbitrap hybrid Fourier transform mass spectrometer (LC-FTMS) was
also reported [60]. It is important to note that the method of analysis
affects the type and quantity of a unique metabolite in the postbiotics.
For example, GC–MS analysis of postbiotics prepared from W. cibaria
showed the presence of oleic and palmitic acids while in HPLC analysis,
only lactic and citric acids were determined at the same postbiotics [61].
4.3. Thin layer chromatography (TLC)
Despite the development of some advanced chromatography tech­
niques, TLC is still used as a routine qualitative test in some laboratories
because it has advantages such as quickness, less tedious, and less costly
over other chromatographic techniques. TLC is the most extensively
studied method for the isolation and structural determination of bio­
surfactants produced by bacterial strains, which are often associated
with a spectroscopic or post chromatographic identification method
[62]. A colorimetric approach using Syldatk, ammonium molybdate or
Ninhydrin reagents (sprayed on the surface of TLC plates) has been
applied to identify moieties, lipid moieties, and amino acids, respec­
tively [62]. In addition, the composition of biosurfactant derived from
Lactobacillus spp. was characterized by TLC followed by post chro­
matographic detection through chromogenic compounds. The results
obtained from TLC confirmed the presence of glycolipids with poly­
saccharides and lipid fractions in postbiotics secreted by Lb. helveticus
[63]. Moreover, two fractions with antibacterial properties were iso­
lated from the postbiotics of Lb. plantarum IMAU10014 using TLC,
consisting of 3-phenylacetic acid, benzeneacetic acid, and 2-propenyl
ester [57].
4.4. Spectrophotometric-based analysis
The spectrophotometric technique can be used to determine the
hydrogen peroxide concentration in postbiotics of selected LAB. The
quantity of total protein in postbiotics of LAB can also be determined by
the colorimetric assay by Bradford method, in which the binding of the
dye (Coomassie Brilliant Blue G-250) to protein cause the sample color
to shift from red to blue (the absorption maximum shift from 465 to
595 nm), being the absorbance changes at 595 nm proportional to the
amount of protein [64]. Furthermore, according to a standard protocol,
the proteinase activity of the postbiotics is evaluated using spectro­
photometric techniques. In this approach, azocasein is used as a chro­
mogenic substrate, and the activity is expressed in terms of absorbance
increase at 440 nm over time [65]. Colorimetric methods have also been
applied to quantify EPS at absorption around 480− 900 nm [66] and
alkaline phosphatase in the postbiotics of LAB [67].
4.5. Nuclear magnetic resonance (NMR) spectroscopy
The NMR is another powerful tool that provides crucial information
about the structure, dynamics, and interactions of biological metabolites
of postbiotics extract. The most common types of NMR are the proton
and carbon-13 NMR spectroscopy which can be applied to any type of
sample containing nuclei possessing spin. In particular, NMR measure­
ment helps to acquire information about postbiotics metabolite from
LAB. Broberg and co-workers showed that postbiotics of Lb. plantarum
comprises (trans, trans)-3,4-dihydroxycyclohexane- 1-carboxylic acid
with antibacterial properties, and they highlighted that they failed to
identify these compounds using another available technique (e.g.,
chromatography) [68]. NMR (1H NMR and 13C NMR) has also been
employed to reveal the presence of phenolic antifungal metabolites
produced by Lb. brevis P68 [69]. Lin and Pan, (2017) utilized 1H NMR
and 13C NMR spectroscopy to confirm the presence of 2-(2− 1-ami­
no-1-hydroxyethoxy) ethyl 2-methylpropanoate as an antimicrobial
substance in the postbiotics of Lb. plantarum NTU 102 [70].
M. Moradi et al.
In another work, the production of benzeneacetic acid and 2-pro­
penyl ester by Lb. plantarum IMAU10014 as an antifungal substance in
the MRS medium were analyzed by 1H NMR and 13C NMR [57]. In
addition, the results of NMR quantification of the biosurfactant derived
from Lb. helveticus, confirmed the presence of several compounds such as
octadecanoic acid as the main lipid consisting of long aliphatic chain
and polysaccharides [63].
4.6. Fourier transform infrared (FTIR) spectroscopy
The FTIR represents a potent characterization technique that has
been recognized in exploration, classification, and identification of
organic and inorganic components. The FTIR technique is a sensitive,
rapid, easy to use, well-established, reagent-less, non-destructive, and
cost-effective approach. Recently, FTIR analysis is recommended to get
qualitative information about metabolites of postbiotics [71]. In this
sense, Shafipour Yordshahi et al. (2020) investigated lyophilized post­
biotics from Lb. plantarum using the FTIR technique. Sharma et al.
(2014) provided a summarized table of the studies that used the FTIR
technique to characterize the biosurfactants [71]. Also, Trabelsi et al.
(2015) monitored the production of EPS during LAB growth using the
FTIR technique [73].
4.7. Other techniques
As stated, postbiotics is a matrix of complex metabolites with diverse
physicochemical and functional properties, so the extraction and/or
isolation step is inevitable since it allows an accurate quantitative and
qualitative assessment of the metabolite. The dialysis is one of the
earliest purification procedure. It seems that the dialysis can help to
distinct low molecular mass antimicrobial metabolites (<1 KDa) from
high molecular mass compounds (>1 KDa) of Lactobacillus spp. [10].
However, the isolation step can be performed by liquid-liquid extraction
(LLE) [58], preparative HPLC [74], TLC [62], solid-phase extraction
cartridge (SPE) [75], QuEChERS methodology [76], and electrophoresis
[77].
Recently, the efficacy of matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) to identify proteinaceous components of
postbiotics from Lactobacillus spp. has received remarkable interest [77,
78]. The application of electrospray ionization mass spectrometry
(ESI-MS) to identify the molecular masses of metabolites produced by
Lactobacillus spp. was reported [75]. Moreover, two-dimensional gel
electrophoresis (2D-PAGE) has also been proposed for the molecular
weight determination [77] in LAB. Finally, some authors used specific
enzymatic kits to quantify acetic and D-/L-lactic acids [78].
5. Factors affecting the chemical composition of postbiotics
5.1. The type of bacterial species
The strain-dependent fermentation of LAB is widely reported.
Organic acid production by LAB is mainly a genus-specific phenomenon
and partially specie-specific [79]. For example, the amount of lactic acid
produced by Lactobacillus spp. and Bifidobacterium spp. is significantly
higher than acetic acid production [80]. Strong positive and negative
correlations were found between lactic and acetic acid contents of
postbiotics and antimicrobial characteristics [80]. Bacterial strains can
also be engineered to secrete unique recombinant metabolites for food,
human, and animal uses [81]. The highest concentration and produc­
tivity of lactic acid were reported in batch and continuous fermentation
of LAB, respectively [82]. Özcelik et al. (2016) investigated organic acid
production ability of LAB in fish infusion broth and MRS broth and
found theproduction of succinic and acetic acids at the highest and
lowest levels, respectively.
8
Enzyme and Microbial Technology 143 (2021) 109722
5.2. Culture type and conditions
The culture composition and incubation conditions can affect me­
tabolites production by LAB [4]. For example, Lactobacillus spp. do not
produce hydrogen peroxide under anaerobic conditions [75]. A time and
cultivation- dependent formation of organic acids by LAB was also re­
ported [83]. The production of organic acids (lactic acid, acetic acid,
succinic acid, and formic acid) by different LAB strains in anchovy
infusion broth is significantly higher than in MRS broth [83]. It was also
found that the postbiotics from different LAB strains prepared in the
low-heat milk revealed significantly higher antifungal activity than
those prepared in milk permeate [39]. In a study, 20 g/L of glucose and
36.20 g/L of yeast extract were used to supplement a modified MRS
medium in order to achieve maximum bacteriocin-inhibitory activity of
postbiotics from Lb. plantarum I-UL4 [84]. The addition of non-ionic
surfactants and emulsifiers, as for instance polysorbate 80, in culture
media can improve the production and release of proteinaceous post­
biotics from Lb. plantarum I-UL4 [85]. Furthermore, supplementation of
Lb. plantarum medium with linoleic acid resulted in the formation of a
therapeutic substance called 10-hydroxy-cis-12-octadecenoic acid
(HYA) [86]. Due to the synergistic activity of postbiotics and prebiotic,
postbiotics of Lb. plantarum prepared in a reconstituted media contain­
ing inulin showed higher antimicrobial activity on a spoilage microor­
ganism (Ped. acidilactici) and various pathogens than the postbiotics
without inulin supplementation [87]. Similarly, in culture media sup­
plemented with glycerol, Lb. Reuteri produced reuterin, a potent anti­
microbial agent [88].
5.3. Co-culturing strategies for postbiotics production
The evidences tend to support the hypothesis that a mixture of multi-
strains is more effective than a mono-strain. The LAB can regulate the
production of specific AAs in postbiotics in the presence of beneficial or
even pathogenic microorganisms, according to a quorum-sensing
mechanism [89]. Co-culturing can facilitate the expression of ribo­
somal synthesized AAs like bacteriocins in LAB [90], also leading to the
discovery of novel AAs. Co-culturing of Lac. lactis subsp. lactis with
Saccharomyces cerevisiae CL01 increased the production of nisin by 85 %
[91]. Enhanced production of nisin (by 50 %) by Lac. lactis subsp. lactis
in the presence of Yarrowia lipolytica was also reported in the sugar beet
molasses [92]. Moreover, Lac. lactis subsp. lactis produce active
nisin-rich postbiotics in co-culturing with harmful pathogens like as
L. monocytogenes and Salm. enterica [89].
5.4. Postbiotics preparation, processing and analysis
For the application of postbiotics in foods, it will be interesting the
use of different forms of presentation, like lyophilized or dried, however,
the processing of extracted postbiotics can influence their composition,
which may decrease the postbiotics activity. For example, lyophilization
promotes the elimination of hydrogen peroxide, which is a factor
responsible for antimicrobial action [93]. The spray drying method also
removes volatile metabolites, as for instance ethanol [75]. There are
evidences suggesting the antagonistic stability of postbiotic metabolites
over time and storage at different conditions [43]. While, with the in­
crease of pH, the antimicrobial performance of postbiotics can nega­
tively be affected [46].
6. Conclusions
This review focused mainly on defining postbiotics and presenting
the methods currently used for preparation and chemical analysis of
postbiotics obtained from LAB. The concept of postbiotics is relatively
new in the food industry and some current applications such as biofilm
removal, biodegradation of food chemical contaminants, and prepara­
tion of nanomaterials using postbiotics have been proposed (Fig. 6). For
M. Moradi et al.
Fig. 6. Different potential food safety application of postbiotics mixture obtained from lactic acid bacteria.
these purposes, it is required to establish standard methods of chemical
analysis. Although several chromatographic (e.g., GC, HPLC, etc.) and
spectrophotometric instrumental methods have been proposed for
postbiotics, identification of some unknown chemical constituents re­
quires high-tech instruments, additionally, for a proper and complete
evaluation, the use of more than one instrument/procedure of analysis
has been preferred.
Moreover, the quantity and quality of metabolites in postbiotics can
be changed by modifying the composition of the culture media. The
brown to brownish-yellow color of the postbiotics prepared with MRS
broth may restrict the postbiotics mixture utilization in some foods,
including many dairy products, especially when using high concentra­
tions. In this case, the individual postbiotic (e.g., bacteriocins, organic
acids, etc.) is generally preferable to postbiotics mixture. Alternatively,
in situ-production of postbiotics by LAB with co-culturing with other
beneficial microorganisms would be an interesting choice.
Despite the facts that favor the use of isolated postbiotic metabolites,
some of them present disadvantages for food application. For example, it
is widely accepted that LAB are associated with the production of
biogenic amines, therefore, it is highly recommended to monitor the
exact chemical content of postbiotics before using it in food products. To
encourage the industrial application and to overcome the main obstacles
of postbiotic preparation and application found in laboratory condi­
tions, such as the high cost and the undesired color appearance, it is
recommended to replace the conventional MRS broth with cost-effective
plant- and animal-based by-products (e.g., whey medium), which is an
area in need of further studies
Author contributions
Mehran Moradi conceived and designed the review. All authors
contributed to the writing of the manuscript.
CRediT authorship contribution statement
Mehran Moradi: Conceptualization, Writing - review & editing.
Rahim Molaei: Writing - review & editing. Jonas T. Guimarães:
Writing - review & editing.
9
Enzyme and Microbial Technology 143 (2021) 109722
Declaration of Competing Interest
The authors reported no declarations of interest.
Acknowledgments
The authors wish to acknowledge Urmia University for support.
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