Accepted Manuscript

Postbiotics: An evolving term within the functional foods field

J.E. Aguilar-Toalá, R. García-Varela, H.S. García, V. Mata-Haro, A.F. González-

Córdova, B. Vallejo-Cordoba, A. Hernández-Mendoza

PII: S0924-2244(17)30276-5
DOI: 10.1016/j.tifs.2018.03.009
Reference: TIFS 2188

To appear in: Trends in Food Science & Technology

Received Date: 10 June 2017

Revised Date: 21 February 2018
Accepted Date: 7 March 2018

Please cite this article as: Aguilar-Toalá, J.E., García-Varela, R., García, H.S., Mata-Haro, V., González-
Córdova, A.F., Vallejo-Cordoba, B., Hernández-Mendoza, A., Postbiotics: An evolving term within the
functional foods field, Trends in Food Science & Technology (2018), doi: 10.1016/j.tifs.2018.03.009.

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 ACCEPTED MANUSCRIPT

1 Postbiotics: An evolving term within the functional foods field

2

3 J.E. Aguilar-Toaláa, R. García-Varelab, H.S. Garcíac, V. Mata-Harod, A.F.

4 González-Córdovaa, B. Vallejo-Cordobaa*, A. Hernández-Mendozaa*

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5

a
6 Laboratorio de Química y Biotecnología de Productos Lácteos, Centro de

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7 Investigación en Alimentación y Desarrollo A.C., carretera a la Victoria KM 0.6.
8 Hermosillo, Sonora 83304. México.

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b
9 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de
10 Jalisco (CIATEJ), Unidad Noreste. Autopista Monterrey-Aeropuerto, Km 10.

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11 Parque PIIT. Vía de Innovación 404, Apodaca, NL 66629. México.
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c
12 Unidad de Investigación y Desarrollo de Alimentos, Instituto Tecnológico de
13 Veracruz, M.A. de Quevedo 2779, Col. Formando Hogar, Veracruz, Veracruz
14 91897. México
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d
15 Laboratorio de Microbiología e Inmunología, Centro de Investigación en
16 Alimentación y Desarrollo A.C., carretera a la Victoria KM 0.6. Hermosillo, Sonora
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17 83304. México.
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18 *Corresponding author:

19 Email: ahernandez@ciad.mx
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20 Email: vallejo@ciad.mx
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21 These authors have contributed equally to the direction of this work

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22

23

24 Revised manuscript submitted to the editor of Trends in Food Science and

25 Technology for publication

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27 Abstract

28 Background: It has been recognized that a number of mechanisms mediating the

29 health benefits of beneficial bacterial cells do require viability. However, new terms
30 such as paraprobiotic or postbiotic have emerged to denote that non-viable

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31 microbial cells, microbial fractions, or cell lysates might also offer physiological
32 benefits to the host by providing additional bioactivity.

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33

34 Scope and Approach: This review provides an overview of the postbiotic concept,

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35 evidence of their health benefits and possible signaling pathways involved in their
36 protective effects, as well as perspectives for applications in foods and

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37 pharmaceuticals. AN
38

39 Key Findings and Conclusions: Postbiotics refers to soluble factors (products or

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40 metabolic byproducts), secreted by live bacteria, or released after bacterial lysis,

41 such as enzymes, peptides, teichoic acids, peptidoglycan-derived muropeptides,
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42 polysaccharides, cell surface proteins, and organic acids. These postbiotics have
43 drawn attention because of their clear chemical structure, safety dose parameters,
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44 long shelf life and the content of various signaling molecules which may have anti-
45 inflammatory, immunomodulatory, anti-obesogenic, antihypertensive,
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46 hypocholesterolemic, anti-proliferative, and antioxidant activities. These properties

47 suggest that postbiotics may contribute, to the improvement of host health by
48 improving specific physiological functions, even though the exact mechanisms
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49 have not been entirely elucidated.

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50

51 Keywords: Bacteria-derived factors; cell lysates; functional foods; health benefits

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52 1. Introduction

53 Several studies have provided plausible evidence of several mechanisms

54 underlying the health-promoting effects of desirable gut bacterial cells; these
55 include modification of the gut microbiota, competitive adherence to mucosa and

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56 epithelium, improvement of epithelial lining barrier function and modulation of the
57 immune system (Bermudez-Brito, Plaza-Díaz, Muñoz-Quezada, Gómez-Llorente,
58 & Gil, 2012; Vyas & Ranganathan, 2012). It is important to note that such

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59 mechanisms are clearly dependent on the viability status of bacteria (Sanders,
60 2009). However, recent evidence suggests that bacterial viability is not necessary

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61 to attain the health-promoting effects, as not all mechanisms nor clinical benefits
62 are directly related to viable bacteria. Research performed by Lee, Zang, Choi,

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63 Shin, and Ji (2002) reported significant immunoregulatory abilities of four different
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64 Bifidobacterium bifidum BGN4 fractions (i.e., whole-cell, cell free extracts, purified
65 cell wall and culture supernatant), where each fraction showed different patterns of
66 immune reactions. Besides, fractions and extracts from Bifidobacterium and
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67 Lactobacillus spp., containing high levels of microbial carbohydrates, have shown

68 profound in vitro tumor-suppressing activities (Choi et al., 2006; Raman, Ambalam,
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69 & Doble, 2016). Therefore, new terms such as paraprobiotic and postbiotic have
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70 emerged which imply that bacterial viability is not an essential requirement for
71 health benefits, providing a potential opportunity in the field of functional foods.
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72 Paraprobiotics, also known as “non-viable probiotics”, “inactivated probiotics” or

73 “ghost probiotics”, refer to inactivated (non-viable) microbial cells, which, when
74 administered in sufficient amounts, confer benefits to consumers (Taverniti &
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75 Guglielmetti, 2011; Tsilingiri & Rescigno, 2013). Despite of proven health benefits
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76 of probiotics, non-viable microbial cells may have safety advantages over

77 probiotics by reducing the risk of microbial translocation, infection or enhanced
78 inflammatory responses, shown for some probiotics in consumers with imbalanced
79 or compromised immune systems (Taverniti & Guglielmetti, 2011). Bacterial cell
80 inactivation may be achieved by physical (mechanical disruption, heat treatment, γ-
81 or UV irradiation, high hydrostatic pressure, freeze-drying, sonication) or chemical
82 (acid deactivation) methods which may alter microbial cell structures or their
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83 physiological functions; hence, bacteria become incapable of growing and

84 therefore retain the beneficial health effects their viable form provides (de Almada,
85 Almada, Martinez, & Sant´Ana, 2016).

86 On the other hand, the term postbiotics, also known as either metabiotics,

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87 biogenics, or simply metabolites/CFS (cell-free supernatants); refers to soluble
88 factors (products or metabolic byproducts) secreted by live bacteria or released
89 after bacterial lysis. These byproducts offer physiological benefits to the host by

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90 providing additional bioactivity (Cicenia et al., 2014; Kostantinov, Kuipers, &
91 Peppelenbosh, 2013; Tsilingiri & Rescigno, 2013). Such soluble factors have been

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92 collected from several bacteria strains; examples include short chain fatty acids
93 (SCFAs), enzymes, peptides, teichoic acids, peptidoglycan-derived muropeptides,

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94 endo- and exo-polysaccharides, cell surface proteins, vitamins, plasmalogens, and
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95 organic acids (Kostantinov et al., 2013; Oberg et al. 2011; Tsilingiri & Rescigno,
96 2013).
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97 Despite the fact that the mechanisms implicated in the health beneficial effects of
98 postbiotics are not fully elucidated, scientific data have provided evidence that
99 postbiotics possess different functional properties including, but not limited to,
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100 antimicrobial, antioxidant, and immunomodulatory. These properties can positively

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101 affect the microbiota homeostasis and/or the host metabolic and signaling
102 pathways, thus affecting specific physiological, immunological, neuro-hormone
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103 biological, regulatory and metabolic reactions (Sharma & Shukla, 2016;
104 Shenderov, 2013).
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105 Currently there is a vast available literature addressing fundamentals, therapeutic

106 and technological aspects of viable “good” bacteria. Most of this literature has
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107 focused on whole cells (alive or heat-killed cells) or its membrane/cell wall
108 components (Sánchez, Ruiz, Guemonde, Ruas-Madiedo, & Margolles, 2012; Reid,
109 2016; Chua, Kwok, Aggarwal, Sun, & Chang, 2017; Huang et al., 2017), and little
110 attention has been paid for the intracellular soluble fraction (so called postbiotics).
111 Although the importance of postbiotics has relatively been overlooked, scientific
112 evidence of their beneficial health effects is progressively increasing (Lee et al.,
113 2004; Kareem, Ling, Chwen, Foong, & Asmara, 2014; Haileselassie et al., 2016;
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114 Nakamura et al., 2016; Tiptiri-Kourpeti et al., 2016; Compare et al., 2017), even
115 though their precise composition and underlying mechanisms are still under
116 investigation. To the best of our knowledge, there are only a few reports
117 summarizing findings on postbiotics, mainly focusing on those from different
118 Lactobacillus species (Shenderov, 2013; Konstantinov et al., 2013; Tsilingiri &

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119 Rescigno, 2013; Patel & Denning, 2013; Cicenia et al., 2014). Hence, this review
120 contributes with new and novel information regarding other bacterial species and

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121 yeast reported as a source of postbiotics, in vitro bioactive properties, in vivo health
122 effects and potential mechanisms involved in different bioactivities. Additionally,

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123 promising analytical tools useful for the detection, identification and quantification
124 of postbiotics, as well as current trends in food and pharmaceutical applications,
will be addressed.

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125 AN
126

127 2. Classes of postbiotics and their characteristics

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128 Gut bacteria depend fully on their host to provide the necessary nutrients that may
129 promote microbiota growth. However, bacteria produce small molecular weight
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130 metabolites during their lifecycle; these compounds play a key role in regulating
131 self-growth, development, reproduction, encourage the growth of other beneficial
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132 organism, cell to cell communication and protection against stress factors (Hibbing
133 Fugua, Parsek, & Peterson, 2010; Netzker et al., 2015; Zhang et al., 2010). Some
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134 of these soluble metabolites may be secreted by live bacteria, or released after
135 bacteria lysis, into the host environment, offering additional physiological benefits
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136 (Figure 1) by modifying cellular processes and metabolic pathways in the host.
137 Although beneficial attributes of one specific bacteria (or a cocktail of bacteria) may
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138 differ from one another, it is theoretically possible, using bioengineering

139 procedures, to design recombinant probiotics capable to exert a variety of
140 beneficial properties by expressing biologically copies of such bioactive
141 metabolites (Chua et al., 2017; Singh, Mal, & Marotta 2017). In general, the
142 postbiotics can be differentiated either by their elemental composition, i.e., lipids
143 (e.g. butyrate, propionate, dimethyl acetyl-derived plasmalogen), proteins (e.g.
144 lactocepin, p40 molecule), carbohydrates (e.g. galactose-rich polysaccharides, and
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145 teichoic acids), vitamins/co-factors (e.g., B-group vitamins), organic acids (e.g.,
146 propionic and 3-phenyllactic acid) and complexes molecules such as
147 peptidoglycan-derived muropeptides, lipoteichoic acids (Kostantinov et al., 2013;
148 Tsilingiri & Rescigno, 2013), or by their physiological functions (See Table 1) which
149 include immunomodulation, anti-inflammatory, hypocholesterolemic, anti-

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150 obesogenic, anti-hypertensive, anti-proliferative, and antioxidant effects (Nakamura
151 et al., 2016; Shin et al., 2010; Sawada et al., 1990).

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152 In general, postbiotics possess several attractive properties such as clear chemical
153 structures, safety dose parameters, and longer shelf life (up to 5 years, when used

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154 as ingredient for foods and beverages or as nutritional supplements) that are
155 greatly sought out (Shigwedha, Sichel, Jia, & Zhang, 2014; Tomar, Anand,

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156 Sharma, Sangwan, & Mandal, 2015). In addition, research performed by
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157 Shenderov (2013) revealed that postbiotics have favorable absorption, metabolism,
158 distribution, and excretion abilities, which could indicate a high capacity to signal
159 different organs and tissues in the host thus eliciting several biological responses.
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160 Moreover, it has been demonstrated that postbiotics can mimic the health effects of
161 probiotics while avoiding the necessary administration of live microorganisms,
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162 which may not always be harmless as previously proved by Tsilingiri et al. (2012)
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163 who found, on a ex vivo assay, that some probiotics can induce a local
164 inflammatory response that resembles the response induced by Salmonella.
165 Furthermore, theoretical concern associated with live probiotic bacteria
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166 administration (e.g., bloating and flatulence, probiotic-related translocation and

167 bacteremia and fungemia, and possible transfer of antibiotic resistance gene) have
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168 been described in case reports, clinical trials and experimental models, in patients
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169 with major (e.g., immunosuppression, premature infants) and minor (e.g.,
170 impairment of the intestinal epithelial barrier, concurrent administration with broad-
171 spectrum antibiotics to which the probiotic is resistant) risk factors for adverse
172 events (Williams, 2010; Doron & Snydman, 2015). Hence, the use of postbiotics
173 may represent a valid and safer alternative to avoids risk linked to live probiotic
174 bacteria, which confer to postbiotics certain practical applicability and functionality

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175 to become a prominent strategy for treating many diseases (Tsilingiri & Rescigno,
176 2013; Haileselassie et al., 2016; Vieira, Fukumori, & Ferreira, 2016).

177

178 3. Methods used to obtain and identify postbiotics

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179 In general, postbiotics have been obtained by using cell disruption techniques,
180 which include heat (Lee et al., 2002; Tejada-Simon & Pestka, 1999), and

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181 enzymatic treatments (Li et al., 2012), solvent extraction (Kim et al., 2011), as well
182 as sonication (Amaretti et al., 2013; Choi et al., 2006; Kim et al., 2006; Matsuguchi

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183 et al., 2003; Ou, Ko, & Lin, 2006; Sharma, Singh, & Kakkar, 2011; Shin et al.,
184 2010; Sokol et al., 2008; Tiptiri-Kourpeti et al., 2016; Zhang et al., 2011).

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185 Besides, additional extraction and clean-up steps such as centrifugation, dialysis,
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186 freeze-dried and column purification have been used to assist obtaining
187 procedures (Matsuguchi et al., 2003; Sawada et al., 1990; Vidal, Donnet-Hugues,
188 Granato, 2002). For instance, Octyl-Sepharose® CL-4B column was used for
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189 isolation of lipoteichoic acid (LTA) from L. johnsonii La1 and L. acidophilus La10
190 (Vidal et al., 2002), and a combination of Macro-prep High Q and Octyl-
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191 Sepharose® CL-4B column for isolation of LTA from L. casei YIT 9029 and L.
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192 fermentum YIT 0159 (Matsuguchi et al., 2003). On the other hand, dialysis against
193 distilled water in a molecular porous membrane tube for 1 d was used to assist the
194 extraction of polysaccharide-glycopeptide complex obtained from L. casei YIT9018
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195 (Sawada et al., 1990); as well as centrifugation in the extraction of intracellular

196 content from various Bifidobacterium spp., Lactobacillus spp., Lactococcus spp.,
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197 and Streptococcus spp. strains (Amaretti et al., 2013; Ou et al., 2006; Zhang et al.,
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198 2011).

199

200 On the other hand, different analytical approaches have been proposed for
201 postbiotic identification. The selection of instrumental technique depends on the
202 analytical goals and the type of characterization (qualitative and/or quantitative)
203 pursued. Accordingly, matrix-assisted laser desorption/ionization time-of-flight

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204 (MALDI-TOF) mass spectrometry has been employed to identify LTA produced by
205 L. plantarum K8 (KCTC10887BP) (Kim et al., 2011); and HPLC and proton nuclear
206 magnetic resonance spectroscopy (1H-NMR) were used to identify and
207 characterize polysaccharide-glycopeptide complexes of Lactobacillus casei
208 YIT9018 (Sawada et al., 1990).

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209 Additionally, chromatography coupled with tandem mass spectrometry and Fourier
210 transform ion cyclotron resonance mass spectrometry with direct infusion, have

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211 been used to identify and characterize metabolites (e.g. fatty acids, glycerolipids,
212 purines, sphingolipids, oligosaccharides) in biological samples (Antunes et al.,

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213 2011; Kok et al., 2013). However, techniques with high efficiency and resolution,
214 low use of solvent and high sensitivity and accuracy such as Ultra Performance

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215 Liquid Chromatography (UPLC) are greatly preferred (Dong & Guillarme, 2013).
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216 Recent studies show that UPLC will be indicated due to superior separation and
217 identification capacity of postbiotics; this technique was used in the profile
218 identification of compounds (e.g. glutathione reductase, amino acid transport
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219 protein) present in intracellular content of L. plantarum (Wang et al., 2016) and the
220 intracellular protein profile (e.g. thioredoxin, phosphoglycerate kinase, cysteine
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221 synthase) in L. mucosea LM1 (Pajarillo, Kim, Lee, Valeriano, & Kang, 2015).
Similarly, Carbon-13 and 1H-NMR, infrared, and electro-spray ionization mass
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222
223 spectrometry techniques have been used to characterize antifungal metabolites
224 (e.g. phenol) produced by L. brevis P68 (Arasu et al., 2015). Moreover, changes in
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225 the protein profile of intracellular content of L. plantarum 423, when exposed to
226 acid conditions (pH 2.5), was determined by gel-free nanoLC-MS/MS proteomics
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227 approach (Heunis, Deane, Smit, & Dicks, 2014). Similarly, the protein profile of L.
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228 plantarum, after bile salt stress, was identified using two-dimensional
229 electrophoresis (2-DE) and liquid chromatography-mass spectrometry analysis
230 (Hamon et al., 2011).

231 Despite that all these techniques could be used to detect, identify and quantify
232 postbiotics, more research about extraction protocols and analytical tools are
233 necessary to allow discovery and characterization of novel postbiotics, but also to
234 understand the mechanisms of action and the signaling pathway modulation.
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235 However, further research is required to optimize media and culture conditions as
236 well as the analytical methods (Anvari, Khayati, & Rostami, 2014). Once laboratory
237 scale optimization is achieved, it must be scaled-up and optimized to ensure
238 maximum postbiotic yield. It is important to note that clinical trials are necessary to
239 define adequate dose and optimal administration frequency (Patel & Denning,

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240 2013). Nevertheless, preclinical studies should be performed prior to initiation of
241 the clinical trial for better selection of the postbiotic candidate. Similar to preclinical

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242 testing of the microbial strains, the details of preclinical activities can vary
243 according to the type of postbiotic and the expected mechanism of action

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244 (Sorokulova, 2008).

245

246 4. Postbiotic bioactivity and/or effects

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247 In recent years, a considerable number of studies using in vitro (e.g., diverse cell
248 lines) and in vivo (e.g., obese and hypertensive rats) models have been used to
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249 assess the potential bioactivity and/or health effects of various postbiotics,
250 including intracellular metabolites and cell wall components, either as isolated
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251 structures or mixtures, such as extracts or suspensions (Robles-Vera et al., 2017).

252 A summary is depicted in Table 1.
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253 Table 1

254 In vitro and in vivo studies of postbiotics, their bioactivity and/or effects.

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Method or tool used for
Bacteria Components Type of study Bioactivity or effect identification or isolation Ref.

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of postbiotic
Bifidobacterium sp.,

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L. acidophilus, L. Cell wall Tejada-Simon
casei, L. delbrueckii components
RAW 264.7 and Pestka,
subsp. bulgaricus, L. and Immunomodulation N.I.
macrophage cell line

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gasseri, L. helveticus, cytoplasmic (1999)
L. reuteri, S. extract

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thermophiles
Faecalibacterium Sokol et al.
Cytosolic
prausnitzii A2-165 Caco-2 cells Immunomodulation N.I.
fraction (2008)

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(DSM 17677)
MALDI-TOF Kim et al.
L. plantarum K8 Lipoteichoic Human monocyte
Immunomodulation Mass spectrometry

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(KCTC10887BP) acids THP-1 cells (2011)
Cell free

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Lee et al.
extracts,
purified cell RAW 264.7 cells Immunomodulation N.I. (2002)
B. bifidum BGN4,
wall and
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supernatant
Octyl-Sepharose® CL- Vidal et al.
L. johnsonni La1, L. Lipoteichoic
Human HT29 cell line Immunomodulation 4B column
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acidophilus La10 acids (2002)

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Macro-prepHigh Q and
L. casei YIT 9029, L. Lipoteichoic RAW 264.7 Octyl-Sepharose® CL- Matsuguchi et
Immunomodulation
fermentum YIT 0159 acids macrophages 4B column al. (2003)

Cell-free Dendritic cells from Mileti, Matteoli,

L. paracasei B21060 Immunomodulation N.I.
supernatants human peripheral IIiev, &

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blood monocytes Rescigno,

(2009)
Cell-free Human mucosa Tsilingiri et al.
L. paracasei B21060 Anti-inflammatory N.I.
supernatants explant of colon (2012)

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Jensen,
Human Immunomodulation Benson,

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Cell wall
Bacillus coagulans polymorphonuclear and anti- N.I.
components Carter, &
cells inflammatory effect

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Endres (2010)
Cell-free Human colonic Cicenia et al.
L. rhamnosus GG Anti-inflammatory N.I.
supernatants smooth muscle cells (2016)

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L. acidophilus (ATCC
HeLa, MCF7, U-87,
43121, ATCC 4356,

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Hep G2, U2OS,
606), L. brevis ATCC Intracellular Choi et al.
PANC-1, HT-29, Antiproliferative N.I.
8287, L. casei (YIT content (2006)
WiDr, DLD-1 and CX-
9029, ATCC 393), L.

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1 cells
rhamnosus GG
Murine CT26 and
Sonicated-cell Tiptiri-Kourpeti

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L. casei ATCC 393 human HT29 colon Antiproliferative N.I.
suspension et al. (2016)
cancer cells line

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Strep. salivarius ssp.
thermophilus ATCC
Ou et al.
19258 and L. Intracellular
In vitro Antioxidant N.I.
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delbrueckii spp. content (2006)
bulgaricus ATCC
11842
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L. acidophilus KCTC
3111, L. jonsonnii
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KCTC 3141, L. Kim et al.

Intracellular
acidophilus KCTC In vitro Antioxidant N.I.
content (2006)
3151, L. brevis KCTC
3498

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L. casei ssp. casei

Zhang et al.
SY13 and L. Intracellular
In vitro Antioxidant N.I.
delbrueckii ssp. content (2011)
bulgaricus LJJ

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7 Bifidobacterium, 11
Lactobacillus, 6 Amaretti et al.
Intracellular

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Lactococcus, and 10 In vitro Antioxidant N.I.
content (2013)
Strep. thermophilus
strains

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Shin et al.
Sonicated-cell High-cholesterol rat Hypocholesterolem
B. longum SPM1207 N.I.
suspension model ic (2010)

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Spontaneously
Polysaccharid HPLC
hypertensive rats and Sawada et al.

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L. casei YIT9018 e-glycopeptide Antihypertensive and
renal hypertensive 1 (1990)
complexes H-NMR
rats models
L. amylovorus Fragmented Nakamura et

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Obese mouse model Antiobesogenic N.I.
CP1563 cells al. (2016)
Dinić et al.
L. fermentum Cell lysate Human hepatoma

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Hepatoprotective N.I.
BGHV110 suspension HepG2 cells (2017)

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Enterococcus lactis
IITRHR1 and Sharma et al.
Intracellular Cultured rat
Lactobacillus Hepatoprotective N.I.
content hepatocytes (2011)
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acidophilus
MTCC447
L. plantarum RG11, Kareem et al.
Cell-free
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RG14, RI11, UL4, In vitro Antimicrobial N.I.

supernatants (2014)
TL1 and RS5
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255 N.I: Not identified; MALDI-TOF: Matrix-assisted laser desorption/ionization time-of-flight; HPLC: High performance liquid
256 chromatography; 1H-NMR: Proton nuclear magnetic resonance spectroscopy;

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257 In the majority of cases, postbiotics are derived from Lactobacillus and
258 Bifidobacterium strains; however, Streptococcus and Faecalibacterium species
259 have also been reported as a source of postbiotics (Kostantinov et al., 2013;
260 Tsilingiri & Rescigno, 2013). It has been proven that supplementation with
261 postbiotics reduces blood pressure which confers the antihypertensive capacity to

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262 these compounds. The mechanism of this protective effect on the endothelial
263 function has not been elucidated; however, this could be due to changes in the gut

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264 microbiota and its metabolic by-products; the restoration of the gut barrier function;
265 and the effects on endotoxemia, inflammation, and renal sympathetic nerve activity

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266 (Robles-Vera et al., 2017).

267 Studies show that the intestinal microbiota also impacts a wide range of functions

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268 in the gastrointestinal tract including the development of the immune system,
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269 defense against pathogens, and inflammation (Klemashevich et al., 2014). With the
270 recent development of the postbiotic concept, growing data, mainly obtained by the
271 analysis of Lactobacilli strains, support the evidence that these beneficial effects
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272 may depend on secreted-derived factors (Compare et al., 2017).

273 Immunomodulation is influenced by retinoic acid-driven mucosal-like dendritic cells

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274 and their subsequent effects on regulatory T-cells in vitro using L. reuteri 17938, by
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275 producing the anti-inflammatory cytokine IL-10 (Haileselassie et al., 2016). In

276 related research, Sokol et al. (2008) reported increased IL-8 levels in Caco-2 cells
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277 when exposed to intracellular extracts and the supernatant fraction of F. prausnitzii.
278 Additionally, the data suggested that administration of cell-free supernatant, in a
279 TNBS-induced colitis mice model, exerted an anti-inflammatory effect by increasing
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280 IL-10 and reducing IL-12, which suggested that secreted metabolites induced this
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281 protective effect. The study also revealed that the anti-inflammatory effect was
282 attributed to a butyrate-independent pathway. Moreover, the proposed mechanism
283 was via NF-кB activation blockade; however, the active molecules involved in this
284 protective effect were not determined. Additional evidence has indicated that the
285 culture supernatant from Lactobacillus paracasei B21060 can protect healthy
286 tissue against the inflammatory properties of invasive Salmonella in a human
287 mucosa explant of colon (Tsilingiri et al., 2012), and that culture supernatant from
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288 Lactobacillus casei DG can mitigate the inflammatory response in ileac and colonic
289 mucosa cultures obtained from post-infectious bowel syndrome patients (Compare
290 et al., 2017).

291 On the other hand, Cicenia et al. (2016) reported that supernatants from

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292 Lactobacillus rhamnosus GG collected at different stages of growth (middle and
293 late exponential, stationary, and overnight) were able to protect human colonic
294 smooth muscle cells (HSMCs) against lipopolysaccharide (LPS)-induced myogenic

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295 damage. Maximal protective effect was observed with supernatants of the late
296 stationary phase, which reverted 84.1% of LPS-induced cell shortening, and

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297 inhibited 85.5% of acetylcholine-induced contraction and 92.7% LPS-induced IL-6
298 secretion.

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Postbiotics have also been described as pathogenic bacteria inhibitors against
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300 pathogens such as Listeria monocytogenes L-MS, Salmonella enterica S-
301 1000, Escherichia coli E-30 and vancomycin-resistant Enterococci when using cell-
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302 free supernatants culture obtained from L. plantarum RG11, RG14, RI11, UL4, TL1
303 and RS5 strains (Kareem et al. 2014). Furthermore, antioxidant activity in in vitro
304 and in vivo models exposed to specific exopolysaccharides (EPS) have been
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305 reported. Xu, Shang, and Li (2011) observed that EPS from Bifidobacterium
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306 animalis RH displayed in vitro inhibition of lipid peroxidation and radical scavenging
307 activity (hydroxyl and superoxide radicals). Moreover, Li et al. (2014) described
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308 that both crude culture extract and purified EPS, from Lactobacillus helveticus
309 MB2-1, exhibited strong scavenging capacity of three kinds of free radicals and
310 chelating capacity to ferrous ion.
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311 Among specific examples of intracellular bacterial enzymes found to have

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312 beneficial health effects (e.g. antioxidant) include glutathione peroxidase (GPx),
313 superoxide dismutase (SOD), nicotinamide adenine dinucleotide (NADH)-oxidase
314 and NADH-peroxidase (Kim et al., 2006; Li et al., 2012). On the other hand, some
315 cell wall components have been associated to in vitro immunomodulatory
316 properties including lipoteichoic acids (LTA), and S-layer proteins (Kostantinov et
317 al., 2013). Additionally, several postbiotics exhibit multiple bioactivities and can
318 simultaneously trigger multiple physiological pathways. For example, some cell
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319 wall components, such as LTA, have been reported to exhibit a variety of
320 bioactivities including antitumor, antioxidant and immunomodulatory capacities
321 (Lebeer, Claes, & Vanderleyden, 2012; Yi, Fu, Li, Gao, & Zhang, 2009). Besides,
322 microbial membrane sterol-like compounds have received special attention
323 including plasmalogens, known as endogenous antioxidants, which confer

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324 resistance to H2O2-induced oxidative stress in several Bifidobacterium strains
325 (Oberg et al., 2011; Oberg, Ward, Steele, & Broadbent, 2012).

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326 According to other works, SCFAs produced by gut microbiota act as signaling
327 molecules improving regulation of lipid metabolism, glucose homeostasis and

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328 insulin sensitivity, through the activation of receptors such as G protein-coupled
329 receptors (GPRs), thus contributing in the regulation of energy balance while

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330 maintaining metabolic homoeostasis (Kimura et al., 2013; Canfora, Jocken, &
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331 Blaak, 2015). Specific SCFAs (e.g. butyrate, acetate and propionate) have also
332 proven to contribute to plasma cholesterol homeostasis in rodents and humans
333 (den Besten et al., 2013).
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334 The hepatoprotective role of postbiotics has also been described. In this sense, cell
335 lysate suspension from Lactobacillus fermentum BGHV110 reduced
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336 acetaminophen-induced hepatotoxicity in HepG2 cells by activating the autophagy

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337 in HepG2 cells through PINK1 signaling pathway (Dinić et al., 2017). In a related
338 work, Sharma et al. (2011) reported the hepatoprotective effect of intracellular
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339 content from Enterococcus lactis IITRHR1 and Lactobacillus acidophilus MTCC447
340 against acetaminophen-induced hepatotoxicity in a cultured rat hepatocytes model.
341 Additionally, the authors found that postbiotics have the potential to restore the
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342 glutathione levels and to reduce levels of oxidative stress biomarkers.

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343 Additionally, postbiotics may also be produced by yeast metabolic activity.

344 Canocini et al. (2011) reported that culture supernatants, obtained from
345 Saccharomyces boulardii, improved wound healing capacity and epithelial cells
346 migration via the activation of α2β1 integrin collagen receptors using in vitro
347 models. Moreover, the authors observed that daily oral administration of such
348 culture supernatant to mice for 7 days improved enterocyte migration along the
349 crypt-villus axis in small intestinal tissues, as examined by immunostaining. The
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350 data suggested that these supernatants could improve the repair process of
351 intestinal epithelium after damage (intestinal restitution) and possess potential
352 therapeutic applications in a wide variety of gastrointestinal disorders.

353 Bioactive properties discovered in postbiotics suggest that these compounds may

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354 contribute in the improvement of host health by providing better specific
355 physiological effects, through the combined effect of postbiotics, other biological
356 metabolites and the live microorganism. This synergy may result in more effective

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357 protective qualities (Thanh, Loh, Foo, Bejo & Azhar, 2010).

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358

359 4.1 Potential mechanism involved in postbiotic bioactivity

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360 Despite the previously stated health beneficial effects of postbiotics, the
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361 mechanisms of action are not fully understood.

362 The protective effect of postbiotics could be caused by compounds that mimic the
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363 beneficial and therapeutic effects of probiotics, even though the mechanisms of
364 action may vary. For instance, hypocholesterolemic mechanisms of probiotic
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365 bacteria, include the inhibition on intestinal cholesterol adsorption and/or the
366 suppression of bile acid re-absorption (Ogawa, Kadooka, Kato, Shirouchi, & Sato,
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367 2014). On the other hand, postbiotics have been reported to activate peroxisome
368 proliferator-activated receptor which causes fatty acid β-oxidation to reduce
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369 triglycerides (Nakamura et al., 2016). Also, postbiotics were found to activate
370 nucleotide-binding oligomerization domain-containing protein 1 that induce cell-
371 autonomous lipolysis in adipocytes (Chi et al., 2014), to decrease the enzyme
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372 activity of hepatic 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS) and 3-

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373 hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and to increase the AMP-

374 activated protein kinase in liver and muscle tissue (den Besten et al., 2013), thus
375 promoting lipid metabolism and dyslipidemia control.

376 Furthermore, it has been found that muramyl dipeptide-based postbiotics may
377 reduce adipose inflammation and glucose intolerance via a nucleotide-binding
378 oligomerization domain-containing protein 2 and though the activation of

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379 transcription factor IRF4 in obese mice (Cavallari et al., 2017). Moreover, this
380 postbiotic reduced hepatic insulin resistance in obesity and low-level endotoxemia.

381 Postbiotics have also proven an antiproliferative specific activity against colon
382 cancer cells; most likely related to the activation of pro-apoptotic cell death

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383 pathways through regulation of immune responses (Tiptiri-Kourpeti et al., 2016). It
384 has been reported that postbiotics obtained from Lactobacillus strains might
385 decrease metalloproteinase-9 activity inhibiting colon cancer invasion (Escamilla,

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386 Lane, & Maitin, 2012). To elucidate the active compound responsible for this effect,
387 the postbiotic (cell-free supernatant) was fractionated based on molecular weight

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388 ranges; it was found that the active inhibitory fraction corresponded to the >100
389 kDa and 50-100 kDa compounds, suggesting that the inhibitory compound may be

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390 a macromolecule such as a protein, nucleic acids, or polysaccharides.
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391 Some studies (Kullisaar et al., 2002; Lin & Chang, 2000; Saide & Gilliland, 2005)
392 determined that cell-free extracts from lactic acid bacteria may exhibit significantly
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393 higher antioxidant capacity than whole cell cultures; suggesting that the antioxidant
394 capacity could be attributed to both enzymatic and non-enzymatic intracellular
395 antioxidants. Furthermore, Bifidobacterium infantis, Bb. breve, Bb. adolescentis,
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396 and Bb. longum are capable of degrading hydrogen peroxide by producing NADH
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397 peroxidase (Shimamura et al., 1992). It has been reported that glutathione
398 peroxidase and glutathione reductase are two important antioxidant enzymes that
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399 protect cells from oxidative damage by scavenging reactive oxygen species (ROS).
400 However, the antioxidant capacity and antioxidant enzyme activity could not be
401 positively correlated for all strains, which indicates that other compounds may be
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402 involved in the antioxidant effect (Kim et al., 2006). In this sense, it has been
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403 proposed that the antioxidant capacity of the intracellular fraction of different strains
404 of Lactobacillus positively correlates with the cellular content of reduced
405 glutathione, an important non-enzymatic antioxidant that plays an essential role in
406 maintaining intracellular redox state (Yoon & Byun, 2004). The antioxidant activity
407 of such non-enzymatic postbiotic could be caused by its ROS and reactive nitrogen
408 species scavenging properties (Amaretti et al., 2013; Zhang et al., 2011).

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409 Additionally, exopolysaccharides have antioxidant activity (Pan & Mei, 2010; Xu et
410 al., 2011). Some studies suggest that this activity is attributed to elevated contents
411 of uronic acid. Some authors (Li et al., 2014; Liu et al., 2011; Xu et al., 2011)
412 proposed that uronic acid plays an important role in the antioxidant properties of
413 polysaccharides from B. animalis RH and L. helveticus MB2-1. Similarly, Li et al.

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414 (2014) reported a polysaccharide from L. helveticus MB2-1, with a greater
415 proportion of negatively charged uronic acid that produced a higher chelating

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416 capacity ferrous ion. Ferrous ions are involved in the formation of free radicals by
417 the Fenton and Haber-Weiss reactions, which generate reactive hydroxyl radicals.

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418 In a related research, a direct relationship between uronic acid content and the
419 radical scavenging capacity of tea polysaccharide was reported (Chen, Zhang, &
Xie, 2004; Chen, Zhang, Qu, & Xie, 2008).

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420 AN
421 It has been suggested that the immunomodulatory and anti-inflammatory
422 capacities of postbiotics are mediated by the inhibition and induction of the immune
423 systems in various animal models. It has been found that postbiotics regulate the
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424 production of the cytokine response and Th1 pathway inhibition (Jensen et al.,
425 2010; Sokol et al., 2008). The antimicrobial activity of postbiotics may be attributed
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426 to the presence of several known and unknown antimicrobial compounds, usually
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427 including but not limited to bacteriocins, enzymes, small molecules, and organic
428 acids, which exhibit bacteriostatic or bactericidal properties against both gram-
429 positive and gram-negative microorganisms (Kareem et al., 2014).
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430 All these properties suggest that postbiotics may contribute to the improvement of
431 the host’s health status by providing better and specific physiological effects,
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432 although the exact mechanisms remain to be elucidated.

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433

434 5. Food and pharmaceutical potential applications of postbiotics

435 The increased knowledge of functional foods has led to the development of a new
436 generation of health products, including those containing probiotics. However, one
437 issue related to the application of probiotics is the occurrence of antibiotic
438 resistance genes in some probiotics strains, as they have the potential to pass the
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439 antibiotic resistance genes to pathogenic bacteria through horizontal gene transfer
440 (Imperial & Ibana, 2016). Another main concern associated with the probiotic
441 product formulations (i.e., pharmaceutical and commercial food-based products) is
442 maintaining bacteria viability during product manufacturing and storage, since the
443 viability of probiotics organism in a delivery system (i.e., pharmaceutical

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444 formulations and commercial food-based products) could be affected by different
445 variables, including interactions with other microbial species present, final acidity of

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446 the product, water activity, temperature, availability of nutrients, growth promoters
447 and inhibitors, inoculation level, fermentation time, dissolved oxygen, and

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448 formulation process procedures such as freeze drying, spray drying or freeze
449 concentration (Shah, 2016). Furthermore, discrepancies between stated and actual
probiotic levels in commercial products for both human and veterinary use have

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450
451 previously been reported (Weese & Martin, 2011). Hence, this lack of probiotic
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452 stability may compromise the expected health benefits provided by probiotic
453 products.
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454 In contrast, postbiotics are supposed to be more stable than the living bacteria they
455 are derived from (Venema, 2013). Phister, O´Sullivan, and McKay (2004) reported
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456 that peptides with antimicrobial properties, namely bacilysin and chlorotetaine,
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457 produced by Bacillus sp. strain CS93 are water soluble and active over a wide pH
458 range, which could allow their application in a wide variety of food products.
459 Furthermore, the use of selected phytase-producing lactic acid bacteria as starters
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460 for breadmaking have been reported as good alternatives for preparing whole
461 wheat bread with low phytate content (Palacios, Haros, Sanz, & Rosell, 2008).
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462 However, previous studies have shown that an advanced hydrolysis of phytate is
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463 achieved by increasing the fermentation time and/or decreasing the pH during
464 whole wheat dough fermentation; conditions that not only may affect the sensory
465 attributes of the final products, but also could influence the synthesis of phytate-
466 degrading enzymes by microorganisms (Haros, Bielecka, Honke, & Sanz 2008).
467 With the use of purified phytate-degrading enzymes, these compounds would not
468 be a serious issue. Another major advantage of postbiotics is their favorable safety
469 profile, as there is no need for the uptake of billions of living microbes (Shigwedha

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470 et al. 2014). Additionally, postbiotics can be applied in a controlled and

471 standardized way, whereas, in the case of the application of living bacteria, the
472 level of the active structure in the intestine is dependent on the number and
473 metabolic activity of the respective strain (Gabriele, 2016). Thus, selected soluble
474 factors from specific bacteria may become a class of bacterial biological strategy

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475 for treating many diseases; however, a big challenge is translating scientific
476 knowledge into commercial applications and thus bridging science and industry.

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477 Currently, cell-free preparations obtained from the metabolic products of different
478 beneficial bacteria have been introduced with potential pharmaceutical applications

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479 in the prevention or treatment of diseases (Klein et al., 2013). For instance,
480 Colibiogen® (Laves-Arzneimittel GmbH, Schötz, Switzerland) a commercially

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481 protein-free filtrate derived from cultures of Escherichia coli (strain Laves 1931),
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482 containing amino acids, peptides, polysaccharides and fatty acids, has been shown
483 to be effective in inhibiting in vitro both antibiotic resistant and sensitive Salmonella
484 isolates (Zihler et al., 2009), in the amelioration of murine colitis (Konrad et al.,
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485 2003), and to significantly reduce skin lesions in patients with polymorphous light
486 eruptions (Przybilla, Heppeler, & Ruzicka, 1989). Hylak® Forte
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487 (Ratiopharm/Merckle GmbH, Germany), a bacteria-free liquid containing metabolic

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488 products (e.g. SCFA, lactic acid an other non-identified metabolites) from E. coli
489 DSM 4087, Streptococcus faecalis DSM 4086, L. acidophilus DSM 414, and L.
490 helveticus DS 4183, has proved to be effective in the management of
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491 salmonellosis in infants (Rudkowski & Bromirska, 1991) and in the treatment of
492 intestinal dysbacteriosis of patients with chronic gastritis (Omarov, Omarova,
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493 Omarova, & Sarsenova, 2014). Besides, has shown to significantly reduce the
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494 incidence and the severity of the radiation-induced diarrhea in radio-oncology

495 patients (Timko, 2010). CytoFlora® (BioRay Inc., Laguna Hills, CA, USA), is a
496 preparation of micronized cell wall lysates of L. rhamnosus, B. bifidum, L.
497 acidophilus, B. infantis, B. longum, S. thermophilus, L. plantarum, L. salivarius, L.
498 reuteri, L. casei, L. bulgaricus, L. acidophilus DDS-1 and Lactobacillus sporogenes,
499 that has been used to correct intestinal dysbiosis, promote a balanced immune
500 response, and improve symptoms in autistic children (Ray, Sherlock, Wilken, &

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501 Woods, 2010). Another commercial product is Del-Immune V® (Pure Research

502 Products, LLC, Boulder, CO, USA), a US Food and Drug Administration-registered
503 formulation containing muramyl peptides, amino acids and DNA fragments of L.
504 rhamnosus V (DV strain), has shown to significantly reduce the severity of
505 gastrointestinal distress in children with Autism Spectrum Disorder when

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506 administrated as blend of Del-Immune V® plus probiotics (West, Roberts, Sichel, &
507 Sichel, 2013).

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508 It has also been reported that probiotic cell lysates may contain hyaluronic acid,
509 sphingomyelinase, lipotechoic acid, exopolysaccharides, peptidoglycan, lactic acid,

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510 acetic acid and/or diacetyl, which provide broad biologic activity that can be
511 harnessed to provide skin benefits such as improving atopic eczema, atopic

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512 dermatitis, healing of burn and scars, skin-rejuvenating properties, improving skin
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513 innate immunity and protecting against photodamage (Lew & Liong, 2013; Kober &
514 Bowe, 2015). According to this, many studies and patents have been published on
515 the use of probiotics extracts to development of personal care products including
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516 oral, underarm (deodorants and antiperspirants) and skin products (Callewaert,
517 Lambert, & van de Wiele, 2017; Coronado-Robles, 2016; Holz et al., 2017; Huang
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518 & Tang, 2015; O´neill & McBain, 2015; Ouwehand, Lahtinen, Tiihonen, 2016).
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519 Although several foods are naturally abundant in postbiotics (e.g., yogurt, kefir,
520 pickled vegetables and kombucha) or their precursors (Chaluvadi, Hotchkiss, &
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521 Yam, 2016), some postbiotics have been intentionally applied to certain foods
522 rather than considering its in situ production by the producer strain. For instance,
523 cell-free supernatant from L. pantarum YML007 has been explored as
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524 biopreservative on soybeans grains (Rather et al., 2013). EPS containing rare
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525 sugars have been investigated for new applications in food industry due to their
526 role in the physicochemical (viscosifying, stabilizing, or water-binding capacities)
527 and sensorial (palatability) characteristics in the final food products; however, with
528 the exception of dextran, EPS from lactic acid bacteria have not yet been
529 commercially exploited as food additives because of their low yields (Torino, de
530 Valdez, & Mozzi, 2015). Nisin, a lantibiotic produced by specific Lactococcus lactis
531 subsp. Lactis strains, is the only bacteriocin approved for use as a food
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532 preservative. Examples of food products with Nisin include canned soups, ice for
533 storing fresh fish, baby foods, baked goods, mayonnaise and dairy products,
534 especially cheeses (Chen & Hoover, 2003). With the above in mind, the use of
535 foods as postbiotics delivery system seems to constitute a field with several
536 opportunities, but also with big challenges.

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537 Improving animal health is another promising field of application, since it has been
538 reported that postbiotics may influence growth performance of hens, broilers and

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539 piglets (Choe, Loh, Foo, Hair-Bejo, & Awis, 2012; Kareem, Loh, Foo, Akit, &
540 Samsudin, 2016a; Loh, Choe, Foo, Sazili, & Bejo, 2014; Loh, Thu, Ling, & Bejo,

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541 2013; Thu, Loh, Foo, Yaakub, & Bejo, 2011). In this sense, a recent research
542 performed by Kareem et al. (2016a) determined that broilers fed with postbiotics

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543 produced by L. plantarum developed significantly higher final body weight and total
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544 weight gain than broilers fed with a basal diet without postbiotics. Also, postbiotics
545 were found to increase significantly duodenal and ileal villus height. Moreover, the
546 combination of postbiotic and inulin may enhance growth performance, improve the
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547 final count of beneficial bacteria and reduce the presence of Enterobacteria and E.
548 coli populations (Kareem, Loh, Foo, Asmara, & Akit, 2016b). In a similar study, Loh
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549 et al. (2014) found a significantly daily higher egg production of hens when treated
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550 with a postbiotic supplement.

551 Some reports proposed that the administration of postbiotics from L. plantarum
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552 exerted a positive effect on growth performance and protein digestibility, as well as
553 reduced diarrhea incidence. Given the collected data, authors suggested that
554 postbiotics altered the mucosal architecture in terms of longer villi and enhanced
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555 animal growth performance. Also, the use of postbiotics modified the intestinal
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556 microbiota, improved the population of protective bacteria (e.g., Lactobacillus and
557 Bifidobacterium) and enhanced the health status of test animals. In addition,
558 postbiotics could contain antimicrobial substances produced by Lactobacillus
559 strains, which may provide nutrients and enhance physiological activities in the
560 animal gut, resulting in absorption improvement and decreased intestinal
561 pathogenic bacteria. Postbiotics can be considered potential contributors as feed
562 additives to achieve higher productivity and better animal health (Loh et al., 2014).
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563 Postbiotics may be useful as microbial-free food supplements, fermented functional

564 foods, and prophylactic drugs, as complementary treatment for several diseases
565 (Chaluvadi et al., 2016). Postbiotics research represents an opportunity not only to
566 understand their mechanisms of action in detail but also to develop new
567 therapeutic strategies for health improvement (Klemashevich et al., 2014;

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568 Shenderov, 2013).

569 On the other hand, the advent of modern techniques for genetic manipulation has

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570 created opportunities for the development of novel bioengineered probiotic strains
571 capable to produce metabolites targeted to the prevention and treatment of several

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572 diseases (Paton, Morona, & Paton, 2012; Sola-Oladokum, Culligan, & Sleator,
573 2017). For instance, genetically modified lactic acid bacteria has been used for

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574 intestinal delivery of antimicrobial peptides (Amalaradjou & Bhunia, 2013),
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575 angiotensin-converting enzyme inhibitory peptides (Yang et al., 2015), cancer-
576 suppressing peptide KiSS1 (Zhang et al., 2016), fusion protein of HSP65 with
577 tandem repeats of P277 (Ma et al., 2014) and glutamic acid decarboxylase and IL-
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578 10 cytokine (Huibregtse et al., 2012; Robert et al., 2014), thus providing promising
579 strategies for the treatment of enteric infections, hypertension, colon carcinoma
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580 and intestinal inflammatory and autoimmune diseases such as Type 1 diabetes
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581 mellitus. Despite of promising biomedical application of recombinant probiotic

582 metabolites, important safety and regulatory aspects still need to be addressed in
583 depth.
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584

585 6. Conclusions
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586 Postbiotics comprise metabolites and/or cell-wall components, secreted by live

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587 bacteria or released after bacterial lysis, with demonstrated beneficial activities in
588 the host. Postbiotics may induce anti-inflammatory, immunomodulatory, anti-
589 obesogenic, anti-hypertensive, hypocholesterolemic, anti-proliferative, and
590 antioxidant activities. These properties suggest that postbiotics may contribute to
591 the improvement of host health by providing specific physiological effects, even
592 though the exact mechanisms have not been fully elucidated. Additional efforts are
593 necessary to allow discovery and characterization of new postbiotics; which may
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594 contribute to the understanding of the signaling pathway modulation. Novel

595 research will allow the generation of detailed information to insure stability during
596 the manufacturing processes of postbiotic products and their efficacy. Special
597 attention should be paid in the development of uniform and stringently defined
598 culture procedures in order to eliminate possible variability of postbiotics

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599 production, since uncontrolled environmental factors can well change metabolism
600 and undergo unexpected transient variability. Beside, well-designed randomized

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601 placebo-controlled human/clinical intervention trials along with metabolomics
602 studies must be conducted looking to support health claims of postbiotics

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603 supplementation.

604

605 Acknowledgement
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606 The authors wish to thank the National Council for Science and Technology
607 (CONACYT) of Mexico for the graduate scholarship for Aguilar-Toalá and for the
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608 research grant CB-2014-01 (230338).

609
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610 References
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1043 Figure caption

1044 Fig. 1. Some postbiotics and their potential local and systemic positive effects in
1045 the host.

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Highlights
• Postbiotics comprise metabolites and/or cell-wall components released by
probiotics
• Postbiotics may, together with probiotics, improve host health
• The exact mechanisms of postbiotic bioactivities have not been fully
elucidated

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