Hindawi

Advanced Gut & Microbiome Research

Volume 2023, Article ID 1984200, 10 pages
https://doi.org/10.1155/2023/1984200

Review Article
The Challenge of Applications of Probiotics in
Gastrointestinal Diseases

William Wolfe,1 Ze Xiang,2 Xi Yu,3 Ping Li,4 Hao Chen,5 Mingfei Yao,2 Yiqiu Fei,2
Yilun Huang,6 Yeshi Yin,7 and Hang Xiao 1
1
Department of Food Science, University of Massachusetts Amherst, MA 01002, USA
2
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases,
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital,
Zhejiang University School of Medicine, Hangzhou 310003, China
3
Faculty of Medicine, Macau University of Science and Technology, Taipa, Macao SAR, China
4
Key Laboratory for Food Microbial Technology of Zhejiang Province, College of Food Science and Biotechnology,
Zhejiang Gongshang University, Hangzhou 310018, China
5
State Key Laboratory of Drug Research, Molecular Imaging Center, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China
6
Alberta Institute, Wenzhou Medical University, China
7
Key Laboratory of Comprehensive Utilization of Advantage Plants Resources in Hunan South, College of Chemistry
and Bioengineering, Hunan University of Science and Engineering, Yongzhou 425100, China

Correspondence should be addressed to Hang Xiao; [email protected]

Received 5 October 2022; Revised 12 November 2022; Accepted 16 November 2022; Published 18 January 2023

Academic Editor: Qixiao Zhai

Copyright © 2023 William Wolfe et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gastrointestinal disease is characterized by gastrointestinal dysfunction with dysbiosis of the microbiome. Probiotics may act as
biological agents in treating gastrointestinal diseases through modifying gut microbiota. However, several challenges, including
safety, stress resistance, postcolonization quantification, and evaluation models, may hinder the application of probiotics in
gastrointestinal diseases. This review introduces the emerging methods for delivering probiotics as well as available materials.
Furthermore, we elucidated bacteriocins and their role in helping probiotics obtain a competitive advantage over other strains
and challenges of large-scale application. Bacteriocins produced by probiotics also showed promising efficacy in gastrointestinal
diseases including the capacity of immune stimulation, intestinal barrier protection, and cytotoxicity against intestinal
tumorigenesis. For the quantification of probiotics in complex microbiomes and evaluation methods of probiotic encapsulated
delivery systems, recent fluorescent labeling technology and various in vitro and in vivo models were also reviewed. Given the
widespread use of probiotic agents in the microecological therapy of gastrointestinal diseases, further understanding of the
multiple challenges of probiotic application and the updated methods to improve the colonization and evaluation system of
probiotics is of great significance for probiotics as live biotherapeutics.

1. Introduction of diseases. Secondly, the minimum viable counts

(~106 CFU/g) are required in order to be beneficial [3].
Although probiotics exhibit high potential as therapeutic However, the most commonly used probiotics usually
agents in treating gastrointestinal diseases, the applications belong to Lactobacillus and Bifidobacterium, which are very
of probiotics are still facing challenges [1]. Firstly, probiotics susceptible to aerobic, and high-temperature environments,
must be safe for human consumption, without any transfer- and the emerging next-generation probiotics demand more
able anti-biotic-resistant genes [2]. Therefore, engineered favorable conditions. Besides, probiotics must endure the
probiotics are usually not allowed to be used for treatment stomach acid and bile during the gastrointestinal transition
2 Advanced Gut & Microbiome Research

[4]. Even though probiotics reach the colon alive, they may The formulation of probiotics for targeted delivery to the
have to adhere to the mucus layer and colonize the colon intestines can be quite challenging in order to resist the
in order to be effective. Therefore, design of probiotic strong acidic gastrointestinal environment. Exposure to gas-
formulations for targeted delivery to the intestines is quite tric acid can be devastating to unprotected probiotics, and
challenging. Bacteriocins could act as a “colonizing peptide,” this is why coating layers and encapsulation techniques must
“killing peptide,” and “signal peptide” by promoting the be applied to protect the probiotics from such strong acidic
colonization of the producing strain in the gut to gain a environment until they are delivered to the targeted place
competitive advantage over other strains. Thirdly, in quanti- [16]. The most commonly used encapsulation approaches
fying probiotics, many problems still need to be solved, such are pH-sensitive and bacteria sensitive coating layers, which
as on-site localization and dynamic monitoring. The existing can release the coated probiotics in the intestine in response
feces examining methods cannot satisfy fast-developing gut to specific pH conditions or certain bacteria colonies [17]
microbiota studies [5]. Therefore, new tools are urgently (Figure 1). Other critical considerations include utilization
needed, such as fluorescent labeling and imaging of gut of natural and economical coating materials; increasing the
microbes. Last but not least, establishment of more proper adhesion of the outer surface of the coating to epithelial cells
in vitro and in vivo models for evaluating the function of of the intestines; enhancing the bioavailability, bile salt
probiotics or probiotic encapsulated delivery systems is hydrolase activity, stability of the probiotics, antagonistic
required. By solving these problems, probiotics may exhibit activity, and efficacy; and targeting capacity of the delivery
good prospects in the food and pharmaceutical fields in the and safety concerns related [10].
future. Here, we discuss the potential therapeutic challenges Various wall materials have been investigated to achieve
of probiotic application and updated recent progress. the goal of targeted delivery of different probiotics, and the
most widely used include dietary fibers, proteins, and natural
polysaccharides [18]. Synthetic materials with good biocom-
2. Targeted Delivery of Probiotics patibility have been used as well. To achieve better efficacy,
combinations of different wall materials have also been
With the ever-growing health needs of people, delivery of attempted, and emulsifiers are added as match makers. More
probiotics through dietary supplements, foods, and bever- importantly, the stability of the probiotics itself has to be
ages has become increasingly popular. Such dietary supple- taken into account, especially during the drying step of food
ments and nutritionally enhanced foods are appealing to processing [19]. Here, the drying techniques usually include
different groups of people with high health care demand, freeze drying, spray drying, extrusion, refractance window
such as women, infants, children, adolescents, the elderly, drying, electrospraying, electrospinning, and emulsifying.
and those recovering from wounds or surgery [6]. The The cost and the temperature of each drying process are
best-known functional foods with added probiotics include important factors to choose a proper processing approach
yogurt, cheese, ice cream, and other dairy products, while [6]. Additionally, advanced techniques such as microfluidics,
novel nondairy products emerged recently, and the market genetic engineering, and 3D printing have also been
keeps expanding [7]. Moreover, novel snacks which have employed to achieve enhanced encapsulation efficiency
been incorporated with desired strain combinations include recently [20].
chocolate bars, cereal, juice, and chips [8].
Successful incorporation of probiotics into dietary sup-
plements or foods requires careful experiments to find out 3. Bacteriocins: Advantages and Challenges
optimum strain combination and formulation to deliver
them. Challenges such as pH and water activity adjustment, Bacteriocins are antimicrobial peptides synthesized in the
temperature control, shelf-life evaluation, and sensory con- ribosome [21], and many probiotic strains were found with
cerns need to be overcome before a perfect product can be a capacity of producing bacteriocins, like the strain Lactococ-
achieved for consumers [9, 10]. Normally, low storage tem- cus lactis which can produce nisin [22, 23]. Bacteriocins are
perature and high-fat content are favorable conditions for classified into four classes according to their molecular
the incorporation of probiotics, which make dairy products weight, chemical structure, thermal stability, and modifica-
ideal candidates for probiotic-enhanced foods. Encapsula- tion [24–26]. It is generally believed that bacteriocins kill
tion and controlled release systems which are popular the targeted cells by destroying the integrity of cell
among pharmacists have been adopted to stabilize the pro- membranes [27], cracking bacterial DNA, interacting with
biotics in certain matrices of foods or beverages recently, intracellular enzymes, and interrupting bacterial protein
resulting in more varieties of snacks containing healthy pro- synthesis [28, 29]. Class I bacteriocins are small posttransla-
biotics [11, 12]. tionally modified ribosomal peptides, which is the class nisin
Most recently, there has been an increased interest in belongs to [30] (Table 1). Class II bacteriocins are small
targeted delivery of probiotics. Normally, such targeted thermostable peptides (5-10 kDa) and nonmodified proteins
delivery is aimed at getting the probiotics to the intestine (such as pediocin PA-1 and plantaricin EF) [31, 32]. Class III
so that better health benefits can be achieved, including bacteriocins are macromolecular heat-sensitive proteins
enhancing gastrointestinal stability, reducing lactose intoler- (>30 kDa) (such as Lactococcin 972) [33]. Class IV bacterio-
ance, relieving diarrhea, boosting immunity, and lowering cins consist of large complexes with carbohydrates or lipid
cholesterol [13–15]. moieties (such as Lactocin 27) [34]. In addition to the above
Advanced Gut & Microbiome Research 3

Delivery of probiotics to the target place by regulating the levels of TNF-α and IL-6 [44], and it also
Coating
protects the epithelial barrier by increasing ZO-1 expression
layers Gastrointestinal stability ↑
in the ileum in obese mice Heeney et al. [31].
Bacteriocins also appear to have selective cytotoxicity
Diarrhea ↓ against colorectal cancer cells in comparison to normal cells
[45], enabling inhibition of intestinal tumorigenesis. In vitro
Immunity ↑ assays revealed that different concentration of nisin inhib-
ited the proliferation of SW48, HT29, and Caco-2 cells to
Lactose intolerance ↓ different degrees [46]. De Giani et al. reported that plantar-
icin P1053 can reduce the proliferation of intestinal cancer
Cholesterol ↓ cells while enhancing the vitality of healthy cells [47]. In
addition to its antitumor activity in vitro, bacteriocins also
Figure 1: Delivery of probiotics to the target place. show good efficacy in vivo studies on tumor inhibition [48,
49]. Reutein is a class II bacteriocin isolated from L. reuteri.
Table 1: Bacteriocin types. Bell et al. found that tumor volume of mice treated with
reutein was significantly reduced when the nude mouse
Bacteriocin model was implanted by HCT116 and SW480 colon cancer
Class I Small posttranslationally modified ribosomal peptides xenograft [45]. Goyert et al. found that reuterin can inhibit
Small thermostable peptides, nonmodified proteins
the growth of colorectal cancer by altering the redox bal-
Class II ance [50].
(5-10 kDa)
Bacteriocins have proved to be an important weapon in
Class III Macromolecular heat-sensitive proteins (>30 kDa)
maintaining microecological balance and gut health [51].
Class IV Large complexes with carbohydrate or lipid moieties However, there are certain challenges in bringing bacterio-
Class V “Kemperman class V” bacteriocin cins into large-scale application. Firstly, bacteriocins
obtained by traditional separation methods such as genetic
engineering to construct recombinant cells have low yields
four class, Kemperman et al. proposed a separate class of and take a long time, while chemical synthesis methods are
cyclic bacteriocins—“Kemperman Class V” bacteriocin [35]. expensive and not suitable for large-scale production [52].
Bacteriocins and bacteriocin-producing probiotics are Furthermore, another issue is the instability of bacteriocins
widespread in the gut. Drissi et al. reported that the Firmi- under low/high pH and pepsin influenced conditions [51].
cutes (mainly Streptococcus and Lactobacillus), which domi- Meanwhile, the degree of expression of bacteriocins under
nate the human gut microbiota, can produce the largest harsh gastrointestinal conditions has not been elaborated,
number of bacteriocin (about 70.79%) [36]. Among them, and further research is required to establish a method to
the vast majority bacteria encoding bacteriocins belong to improve the stability and efficacy of bacteriocin as biologics.
lactic acid bacteria (LAB), indicating that bacteriocins from
LAB play a crucial role in maintaining the balance of gut 4. Quantification of Probiotics:
microflora. Bacteriocins could act as a “colonizing peptide,” Fluorescence Labeling
“killing peptide,” and “signal peptide” by promoting the
colonization of the producing strain in the gut to gain a At present, the identification and quantification of most gas-
competitive advantage over other strains [23], targeting trointestinal microbiomes are highly dependent on the high-
undesirable pathogens without negatively affecting beneficial throughput DNA sequencing of fecal microflora [53]. The
flora [37], and participating in quorum sensing with other gut microbiota is composed in a certain proportion to form
bacteria or to signaling cells in the host immune system, a stable ecosystem, where bacteria cooperate and restrain
respectively. Pediocin PA-1, produced by Pediococcus each other. It is controversial whether the fecal microbiome
acidilactici UL5, was reported to reduce the intestinal coloni- can represent the whole composition of the colonized bacte-
zation of vancomycin-resistant enterococci (VRE) in vivo ria in the gut [54, 55]. Simultaneously, many problems still
[38]. Garvicin ML, produced by L. garvieae DCC43, was able need to be solved in quantifying probiotics, such as on-site
to improve host health by modifying intestinal microbiota, localization and dynamic monitoring. Many foreign bacteria
including potentially problematic bacteria inhibition, LAB are excreted with the feces as they cannot colonize in the
proportion increase, and triglyceride level decrease [23]. intestinal tract. The composition of bacteria in feces exceeds
Bacteriocins are involved in immune regulation and that of colonized gut microbiota. The existing feces examin-
intestinal epithelial barrier protection, thus controlling dis- ing methods cannot satisfy rapidly developing gut microbi-
eases such as obesity (Table 2) [39, 40]. Nisin and CBP22 ota studies. Therefore, new tools are urgently needed to
were found to protect the host from pathogens’ infection detect microbial communities and their presence in the
by enhancing the intestinal immunity [41, 42]. Nisin could digestive tract. Gut microbes’ fluorescent labeling and imag-
also affect neutrophils and induce the formation of neutro- ing could be an appropriate method to resolve this problem.
phil extracellular traps in vitro in a dose-dependent manner Researchers have recently been devoted to developing
[41, 43]. Yin et al. reported that plantaricin EF can improve convenient bacteria fluorescent imaging methodologies.
the symptoms of acute inflammatory bowel disease in mice Transferring fluorescent protein genes into bacteria genes
4 Advanced Gut & Microbiome Research

Table 2: Effect of bacteriocins on gastrointestinal health.

Bacteriocin Class Producing strain Effect References

Abp118 II L. salivarius UCC118 Alleviate metabolic abnormalities associated with obesity [39]
Reduce the intestinal colonization of VRE; inhibit the growth of
Pediocin PA-1 II P. acidilactici PAC1.0 [38, 49, 96]
DLD-1 and HT29
Significantly increase the counts of total LAB and decrease the
Garvicin ML II L. garvieae DCC43 [23]
blood serum levels of triglycerides
Nisin I L. lactis Regulate the intestinal immune; inhibit colorectal cancer in vitro [43, 97, 98]
Prevention of LPS-induced gut barrier dysfunction by modulating
CBP22 I Clostridium butyricum ZJU-F1 [42]
the immune system
Plantaricin P1053 I L. plantarum PBS067 Reduce proliferation of cancer-causing human intestinal cells [47]
Anticancer activity against HCT-116, SW480, RKO, and DLD-1
Reuterin II L. reuteri [45, 50]
colorectal cancer cells
Nisin A I L. lactis Inhibit colorectal cancer cells LS180, SW48, HT29, and Caco-2 [99]
Pediocin K2a2-3 II P. acidilactici K2a2-3 Inhibit the proliferation of HT29 [100, 101]
Alter the proportions of several important gut bacteria, such as
Bactofencin A II L. salivarius DPC6502 [102]
Fusobacterium, Bacteroides, and Bifidobacterium
Ameliorate the effects of obesogenic diets; acute inflammatory
Plantaricin EF II L. plantarum 163 [31, 44]
bowel disease
Gassericin A V L. gasseri LA39 Affect the differentiation and development of adipocytes [103]

was initially attempted [56, 57]. However, most of the gut if the bacterial metabolic pathways changed, which is likely
microbiota is hard to isolate and culture under artificial con- to happen in gut microbiota.
ditions, making the gene transfer challenging [58]. Thus, Due to negative surface charges, different metal cation
in vitro testing tools based on bacterial nucleic acid sequenc- sterilants are used [68]. Researchers also utilize the contrast
ing, such as fluorescence in situ hybridization (FISH), were reagent labeled metal cations for bacteria imaging. The zin-
adopted [59]. It is unfortunate that FISH can only label dead c(II)-coordinated compound is combined with the near-
bacteria. Therefore, many attempts have been made to label infrared fluorescence group, and the probe has high specificity
bacteria in vivo. for wounds infected by Staphylococcus aureus [69]. Combin-
Since some antibiotics can specifically bind to the bacte- ing antimicrobial peptide G3KL with fluorescent dyes can tar-
rial outer membrane, antibiotics are conjugated with fluores- get Gram-negative bacteria and accumulate on the cell
cence dyes to form bacterial targeting probes, which can membrane [70]. AIE material TBP-1 could target the cell
selectively label bacteria in complex samples [60–62]. The membrane of methicillin-resistant Staphylococcus with no
combination of lipopolysaccharide-targeted polymyxin B drug resistance [71]. These probes have a simple chemical
and Cy3 dye can form a Gram-negative bacterial-specific structures which are easy to synthesize, but they still face the
probe [61]. The peptidoglycan-targeted vancomycin Cy3 same safety concern as antibiotic-based probes [72]. Positive
conjugation probe could label Gram-positive bacteria after charged dye MitoTracker Red is adopted in bacteria in vitro
incubation with intestinal flora [62]. The most significant fluorescent imaging [73]. However, the cost is expensive due
concern of those probes is the toxicity. Even low concentra- to the large amount needed for in vivo labeling by intragastric
tions of antibiotic-based imaging probes could damage bac- administration. On the other hand, some cation probes like
teria, resulting in drug resistance and host-microbiota cationic peptides could easily penetrate the mammalian cells,
disorder [63, 64]. resulting in high background noise [74, 75].
Consequently, the metabolic labeling strategy is pro- In summary, despite deficiencies and shortcomings,
posed to solve this problem. Metabolic-based mimic probes fluorescence imaging technology is highly valued in probi-
such as unnatural precursors or substrates could tag bacteria otic quantification. The advantages of high sensitivity, accu-
during proliferation or energy harvesting. And it has been rate resolution, and low cost make it widely used in bacterial
applied to track gut microbiota colonization and spatial dis- quantification and imaging. Better fluorescent probes and
tribution [65]. The near-infrared sulfide quantum dots (PbS labeling strategies are eagerly needed to solve all the above
QD) coated with ribonuclease A (RNase-A) could be concerns.
assembled with bacterial surface proteins [66]. The filamen-
tous temperature-sensitive protein (FtsZ) inhibitor oxazol- 5. In Vitro and In Vivo Models
benzamide is attached to a fluorophore that can mark
Staphylococcus aureus, Enterococcus faecalis, Streptococcus 5.1. Bacterial Growth Inhibition Models. Escherichia coli,
pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Salmonella, Listeria, Clostridium difficile, Helicobacter pylori,
Pseudomonas aeruginosa [67]. But this strategy would fail and Candida albicans are common pathogens that cause
Advanced Gut & Microbiome Research 5

gastrointestinal diseases. A large number of studies have gut microbiota activity and community composition using
used the traditional in vitro determination of the growth the SHIME, and they found that during treatment, Akkerman-
inhibition of probiotics on pathogenic bacteria to screen sia muciniphila, Bifidobacteria spp., and Firmicutes increased
and assess the potential efficacy of probiotics (Figure 2). while Lactobacillus spp. and Bacteroidetes decreased [85].
Abishad et al. investigated the effects of Lactobacillus aci- Due to the limited studies, the usability and feasibility of
dophilus on a multi-drug-resistant enteroaggregative Escher- in vitro intestinal microbiota simulation models need to be
ichia coli (MDR-EAEC) strain, and they proved potential further investigated.
antibacterial and antibiofilm activity of green synthesized
silver nanoparticles against MDR-EAEC strains with antiox-
5.3. Cell and Organoid Models. There are different models
idant properties [76]. Ruiz et al. used the synbiotic combina-
available that mimic the human intestinal epithelium and
tion of Bifidobacterium longum subsp. infantis CECT7210
are thus available for studying probiotic and pathogen inter-
and oligosaccharides to examine the antimicrobial activity
actions in the gastrointestinal tract. Standard 2D models are
against Escherichia coli, Cronobacter sakazakii, Listeria
comprised of culture plates as well as Transwell inserts, and
monocytogenes, and Clostridium difficile in coculture experi-
newer 3D models like organoids, enteroids, and organ-on-a-
ments, and they found that the new symbiotic may be an
chip have been built to assess probiotic-pathogen interac-
effective supplement for infant health [77]. Cizeikiene and
tions [86]. Chen et al. found that the inhibition of H. pylori
Jagelaviciute evaluated the antibacterial activities of twelve
adhesion and the invasion of gastric epithelial cells and
pathogenic strains belonging to Staphylococcus aureus,
interleukin-8 production were significantly decreased by treat-
Escherichia coli, Staphylococcus chromogenes, and Staphylo-
ment with the Lactobacillus strains by using an in vitro cell-
coccus hyicus species by performing agar diffusion assay
based model [87]. Wei et al. proved that Bifidobacterium
and broth inhibition assay methods. The results demon-
longum JDM301 partially relieved damage to tissues caused
strated that Lactobacillus acidophilus DSM 20079, Bifidobac-
by C. difficile and also decreased the number of C. difficile
terium pseudolongum DSM 20099, and Bifidobacterium
and toxin levels by using in vitro cell models [79]. Engevik
animalis DSM 20105 can serve as probiotic candidates [78].
et al. assessed the role of Lactobacillus reuteri in modulating
Except for detecting the growth of pathogens, pathogenic
the host immune system in an organoid-dendritic coculture
genes or genes related to host infection were also utilized for
and demonstrated that both L. reuteri secreted factors and its
screening probiotics. Wei et al. found that Bifidobacterium
bacterial components are able to promote dendritic cell
longum JDM301 not only played important roles in the
maturation [88]. In addition, some ex vivo models, like the
growth inhibition against C. difficile but also directly pro-
Microbiota-human Intestine on chip (MihI-oC) [89], the
moted the degradation of clostridial toxin [79]. Ghadimi
Ussing chamber [90], and the human intestinal in vitro organ
et al. evaluated the probiotic effects of Bifidobacterium ani-
culture (IVOC) model [91], also have been developed. Efforts
malis R101-8 by detecting the expression levels of key lipid
are under way to develop broader systems that connect
metabolism genes, inflammation-related cytokines, and bio-
multiple organotypic models to ultimately converge into a
markers, and they demonstrated that B. animalis R101-8 can
“body-on-a-chip” [92].
improve biomarkers of metainflammation through the
molecular/signaling mechanisms triggered by proinflamma-
tory bacteria and lipids [80]. Additionally, with the rapid 5.4. Animal Models. Animal models provide very controlled
development of big data science and bioinformatics, some environments and enable the use of germ-free animals for
silicon models have also been developed for probiotic func- studying the interactions between host and microbe as well
tion evaluation. Mathematical models and genome scale as potential pathogens. In addition, animal models provide
metabolic models have been used for predicting and evaluat- the possibility to collect samples from different parts of the
ing some bacterial probiotic functions [81, 82]. gastrointestinal tract that is not possible within clinical trials.
Although mice and rats are most frequently used, Caenor-
5.2. In Vitro Intestinal Microbiota Simulation Models. A habditis elegans, honey bees, Ciona robusta, fruit flies, and
more recent batch fermentation method simulating the dis- greater wax moths also have been developed for assessing
tal colon could potentially be used for studying probiotic probiotic-pathogen interactions [86]. Chen et al. indicated
pathogen interactions. Models like SHIME, SIMGI, TIM-2, that colonization of H. pylori and induced stomach inflam-
ECSIM, EnteroMix, and PolyFermS have been built for the mation were alleviated by Lactobacillus strains [87]. Scalfaro
human intestinal microbiota simulation [83]. Although most et al. used G. mellonella to evaluate the antibacterial activity
of these models were used for investigating the interactions of L. rhamnosus GG and Clostridium butyricum Miyairi
between dietary functional factors or drugs and intestinal against three enteric pathogens causing infections, and these
microbiota, the effect of probiotics, prebiotics, and synbiotics results suggested that G. mellonella larvae are a potentially
on the gut microbiota has been investigated by using these useful in vivo model, which can complement in vitro assays
models. Duque et al. found that the probiotic, prebiotic, to prescreen candidate probiotics [93]. Although genome
and synbiotic treatments resulted in a positive modulation editing technology has made rapid progress and many
of the gut microbiota and metabolic activity of children with genome editing rats and mice have been established, there
autism spectrum disorder by using SHIME [84]. Marzorati are still few cases of genome editing animals used in
et al. investigated the effects of MegaSporeBiotic™ (an oral, probiotic-related research. Hence, more studies on animal
spore-based probiotic comprised of five Bacillus spp.) on models are needed.
6 Advanced Gut & Microbiome Research

Three methods to screen and assess the potential efficacy of probiotics

In vitro determination of the growth inhibition of probiotics on

pathogenic bacteria

Utilizing pathogenic genes or genes related to host infection

Mathematical models and genome scale metabolic models

Figure 2: Three methods to screen and assess the potential efficacy of probiotics.

5.5. Human Clinical Experiments. Regardless of the fact that Conflicts of Interest
in vitro models and animal experiments have the advantages
of simple operation, controllable experimental factors, and The authors declare no competing interests.
low research cost, reliable evidence of the impact of probio-
tics on human health still largely depends on human clinical Authors’ Contributions
experiments. Numerous studies have also further promoted
the development and sales of probiotic products through Hang Xiao and William Wolfe developed the idea, and Wil-
human clinical trials. For example, E. coli includes a variety liam Wolfe drafted the manuscript. Ze Xiang, Xi Yu, and
of strains, most of which are regarded as opportunistic Ping Li helped with the table and figures. Hao Chen, Mingfei
pathogens. Human clinical trials are one of the important Yao, Yiqiu Fei, and Yilun Huang revised the manuscript.
reasons why Escherichia coli Nissle 1917 is recognized as a Yeshi Yin developed the idea and drafted the outline. Hang
probiotic and widely accepted and used [94]. Dronkers Xiao organized, reviewed and finalized the manuscript. All
et al. performed the global analysis of clinical trials with pro- authors contributed to the manuscript and approved for
biotics, and they found that L. rhamnosus GG and Bifidobac- publication.
terium animalis ssp. lactis BB12 are the most widely studied
probiotic strains [95]. Although there exist several clinical Acknowledgments
trials to investigate the benefits of probiotics in gastrointesti-
nal diseases, the results can be inconsistent and sometimes This work was supported in part by the National Key Research
contrary, which may cause by many factors, including trail and Development Program of China (2021YFA1301100), the
design, group size, group characteristics, and dosage. Hence, Natural Science Foundation of China (32001683 and
the design of human clinical experiments should consider 81790631), the Research Project of Jinan Microecological Bio-
probiotics, host population, and study design carefully. medicine Shandong Laboratory (JNL-2022001A), and the
National Institutes of Health (R01AT010229).
6. Conclusions
References
Although probiotics can serve as therapeutic agents in treat-
ing gastrointestinal diseases, there are still several challenges, [1] H. Shen, Z. Zhao, Z. Zhao, Y. Chen, and L. Zhang, “Native
which may limit the effective applications. Understanding and engineered probiotics: promising agents against related
the existing challenges will make better use of probiotics in systemic and intestinal diseases,” International Journal of
gastrointestinal diseases. Importantly, if probiotics cannot Molecular Sciences, vol. 23, no. 2, p. 594, 2022.
be delivered to the targeted place, the anticipated effect will [2] M. Sanders, D. Merenstein, C. Merrifield, and R. Hutkins,
not be achieved. Bacteriocins and bacteriocin-producing “Probiotics for human use,” Nutrition bulletin, vol. 43,
no. 3, pp. 212–225, 2018.
probiotics can maintain microecological balance and gut
health, whereas troubles such as large-scale production and [3] R. Guevarra and V. L. Barraquio, “Viable counts of lactic acid
bacteria in Philippine commercial yogurts,” International
instability under the certain environments limit their further
Journal of Dairy Science & Processing, vol. 2, no. 5, pp. 24–
application. For probiotic quantification, fluorescence imag- 28, 2015.
ing technology has been concerned, and better fluorescent
[4] C. Picard, J. Fioramonti, A. Francois, T. Robinson, F. Neant,
probes and labeling strategies are required to solve the exist- and C. Matuchansky, “Review article: bifidobacteria as probi-
ing drawbacks. Up to now, several in vitro and in vivo otic agents – physiological effects and clinical benefits,” Ali-
models have been developed to assess the potential efficacy mentary Pharmacology & Therapeutics, vol. 22, no. 6,
of probiotic, including bacterial growth inhibition models, pp. 495–512, 2005.
in vitro intestinal microbiota simulation models, cell and [5] R. Choudhary and R. Mahadevan, “Toward a systematic
organoid models, animal models, and human clinical exper- design of smart probiotics,” Current Opinion in Biotechnol-
iments. Nevertheless, the development of these models is ogy, vol. 64, pp. 199–209, 2020.
still in the initial stage, and more efficient and proper models [6] S. Razavi, S. Janfaza, N. Tasnim, D. L. Gibson, and M. Hoorfar,
need to be established. “Microencapsulating polymers for probiotics delivery systems:
Advanced Gut & Microbiome Research 7

preparation, characterization, and applications,” Food Hydro- [22] O. Gillor, A. Etzion, and M. A. Riley, “The dual role of bacte-
colloids, vol. 120, no. 10, article 106882, 2021. riocins as anti- and probiotics,” Applied Microbiology and
[7] C. González-Ferrero, J. M. Irache, and C. González-Navarro, Biotechnology, vol. 81, no. 4, pp. 591–606, 2008.
“Soybean protein-based microparticles for oral delivery of [23] Ö. C. O. Umu, C. Bäuerl, M. Oostindjer et al., “The potential
probiotics with improved stability during storage and gut of class II bacteriocins to modify gut microbiota to improve
resistance,” Food Chemistry, vol. 239, pp. 879–888, 2018. host health,” PLoS One, vol. 11, no. 10, article e0164036, 2016.
[8] H. J. Chen, D. A. Lin, F. Liu et al., “Transdermal Delivery of [24] Y. Cui, L. Luo, X. Wang et al., “Mining, heterologous expres-
Living and Biofunctional Probiotics through Dissolvable sion, purification, antibactericidal mechanism, and application
Microneedle Patches,” ACS Applied Bio Materials, vol. 1, of bacteriocins: a review,” Comprehensive Reviews in Food
no. 2, pp. 374–381, 2018. Science and Food Safety, vol. 20, no. 1, pp. 863–899, 2021.
[9] A. Gani, A. Shah, M. Ahmad, B. A. Ashwar, and F. A. [25] E. M. Johnson, D. Y.-G. Jung, D. Y.-Y. Jin, D. R. Jayabalan, D. S.
Masoodi, “β-d-glucan as an enteric delivery vehicle for pro- H. Yang, and J. W. Suh, “Bacteriocins as food preservatives:
biotics,” International Journal of Biological Macromolecules, challenges and emerging horizons,” Critical Reviews in Food
vol. 106, pp. 864–869, 2018. Science and Nutrition, vol. 58, no. 16, pp. 2743–2767, 2018.
[10] K. Kailasapathy, J. Versalovic, and M. Wilson, “Formulation, [26] R. Kumariya, A. K. Garsa, Y. S. Rajput, S. K. Sood, N. Akhtar,
Administration, and Delivery of Probiotics,” in Therapeutic and S. Patel, “Bacteriocins: classification, synthesis, mecha-
Microbiology: Probiotics and Related Strategies, Wiley, 2014. nism of action and resistance development in food spoilage
[11] A. C. Anselmo, K. J. Mchugh, J. Webster, R. Langer, and causing bacteria,” Microbial Pathogenesis, vol. 128, pp. 171–
A. Jaklenec, “Biomaterials: layer-by-layer encapsulation of 177, 2019.
probiotics for delivery to the microbiome (Adv. Mater. 43/ [27] P. D. Cotter, R. P. Ross, and C. Hill, “Bacteriocins – a viable
2016),” Advanced Materials, vol. 28, no. 43, pp. 9442–9442, alternative to antibiotics?,” Nature Reviews Microbiology,
2016. vol. 11, no. 2, pp. 95–105, 2013.
[12] C. G. Olnood, S. S. M. Beski, P. A. Iji, and M. Choct, “Delivery [28] S. Heu, J. Oh, Y. Kang et al., “gly gene cloning and expression
Routes for Probiotics: Effects on Broiler Performance, Intesti- and purification of glycinecin A, a bacteriocin produced by
nal Morphology and Gut Microflora,” vol. 1, Tech. Rep. 3, Xanthomonas campestris pv. glycines 8ra,” Applied and Envi-
Animal Nutrition, 2015. ronmental Microbiology, vol. 67, no. 9, pp. 4105–4110, 2001.
[13] R. Gheorghita, L. Anchidin-Norocel, R. Filip, M. Dimian, and [29] S. D. Todorov, B. D. G. de Melo Franco, and J. R. Tagg, “Bac-
M. Covasa, “Applications of biopolymers for drugs and pro- teriocins of gram-positive bacteria having activity spectra
biotics delivery,” Polymers, vol. 13, no. 16, p. 2729, 2021. extending beyond closely-related species,” Beneficial Microbes,
[14] J. Kim, S. P. Hlaing, J. Lee, A. Saparbayeva, and J. W. Yoo, vol. 10, no. 3, pp. 315–328, 2019.
“Exfoliated bentonite/alginate nanocomposite hydrogel [30] G. G. F. Newton, E. P. Abraham, and N. J. Berridge, “Sulphur-
enhances intestinal delivery of probiotics by resistance to gas- containing amino-acids of nisin,” Nature, vol. 171, no. 4353,
tric pH and on-demand disintegration,” Carbohydrate Poly- p. 606, 1953.
mers, vol. 272, no. 42, article 118462, 2021. [31] D. D. Heeney, Z. Zhai, Z. Bendiks et al., “Lactobacillus plan-
[15] L. I. Li-Juan and S. F. School, “Effects of dietary chitosan and tarum bacteriocin is associated with intestinal and systemic
probiotics on growth and non-specific immunity of Pelteoba- improvements in diet-induced obese mice and maintains epi-
grus fulvidraco,” Journal of Anhui Agricultural Ences, vol. 26, thelial barrier integrity in vitro,” Gut Microbes, vol. 10, no. 3,
no. 6, pp. 2614–2619, 2013. pp. 382–397, 2019.
[16] L. P. Ta, E. Bujna, S. Kun, D. Charalampopoulos, and V. V. [32] T. M. Kuniyoshi, P. M. O'Connor, E. Lawton et al., “An oxi-
Khutoryanskiy, “Electrosprayed mucoadhesive alginate- dation resistant pediocin PA-1 derivative and penocin A dis-
chitosan microcapsules for gastrointestinal delivery of probio- play effective anti-listeria activity in a model human gut
tics,” International Journal of Pharmaceutics, vol. 597, article environment,” Gut Microbes, vol. 14, no. 1, 2022.
120342, 2021. [33] C. Madera, P. García, A. Rodríguez, J. E. Suárez, and
[17] C. C. Dodoo, J. Wang, A. W. Basit, P. Stapleton, and B. Martínez, “Prophage induction in Lactococcus lactis by
S. Gaisford, “Targeted delivery of probiotics to enhance gastro- the bacteriocin Lactococcin 972,” International Journal of
intestinal stability and intestinal colonisation,” International Food Microbiology, vol. 129, no. 1, pp. 99–102, 2009.
Journal of Pharmaceutics, vol. 530, no. 1-2, pp. 224–229, 2017. [34] G. C. Upreti and R. D. Hinsdill, “Production and mode of
[18] C. Iyer, “Studies on Co-Encapsulation of Probiotics and Pre- action of lactocin 27: bacteriocin from a homofermentative
biotics and Its Efficacy in Survival, Delivery, Release and Lactobacillus,” Antimicrobial Agents and Chemotherapy,
Immunomodulatory Activity in the Host Intestine, [Ph.D. vol. 7, no. 2, pp. 139–145, 1975.
thesis],” University of Western Sydney, Australia, 2005. [35] R. Kemperman, A. Kuipers, H. Karsens, A. Nauta,
[19] P. Devani, A. Ruparelia, V. Garcia-Larsen et al., “Optimal O. Kuipers, and J. Kok, “Identification and characterization
Mode of Delivery for Using Probiotics or Prebiotics to Pre- of two novel clostridial bacteriocins, circularin A and closti-
vent Eczema: A Systematic Review and Meta-Analysis,” Clin- cin 574,” Applied and Environmental Microbiology, vol. 69,
ical and Experimental Allergy, vol. 47, no. 12, p. 1700, 2017. no. 3, pp. 1589–1597, 2003.
[20] N. Kevin, A. Nancy, R. Patricia et al., “High-efficiency delivery of [36] F. Drissi, S. Buffet, D. Raoult, and V. Merhej, “Common
CRISPR-Cas9 by engineered probiotics enables precise micro- occurrence of antibacterial agents in human intestinal micro-
biome editing,” Molecular Systems Biology, vol. 17, no. 10, 2021. biota,” Frontiers in Microbiology, vol. 6, p. 441, 2015.
[21] P. D. Cotter, C. Hill, and R. P. Ross, “Bacteriocins: developing [37] J. W. Hegarty, C. M. Guinane, R. P. Ross, C. Hill, and P. D.
innate immunity for food,” Nature Reviews Microbiology, Cotter, “Bacteriocin production: a relatively unharnessed
vol. 3, no. 10, pp. 777–788, 2005. probiotic trait?,” F1000Research, vol. 5, p. 2587, 2016.
8 Advanced Gut & Microbiome Research

[38] M. Millette, G. Cornut, C. Dupont, F. Shareck, [53] J. Qin, M. H. I. T. Consortium, R. Li et al., “A human gut
D. Archambault, and M. Lacroix, “Capacity of human microbial gene catalogue established by metagenomic
nisin- and pediocin-producing lactic acid bacteria to reduce sequencing,” Nature, vol. 464, no. 7285, pp. 59–65, 2010.
intestinal colonization by vancomycin-resistant enterococci,” [54] G. P. Donaldson, S. M. Lee, and S. K. Mazmanian, “Gut bio-
Applied and Environmental Microbiology, vol. 74, no. 7, geography of the bacterial microbiota,” Nature Reviews.
pp. 1997–2003, 2008. Microbiology, vol. 14, no. 1, pp. 20–32, 2016.
[39] E. F. Murphy, P. D. Cotter, A. Hogan et al., “Divergent met- [55] C. Tropini, K. A. Earle, K. C. Huang, and J. L. Sonnenburg,
abolic outcomes arising from targeted manipulation of the “The gut microbiome: connecting spatial organization to
gut microbiota in diet-induced obesity,” Gut, vol. 62, no. 2, function,” Cell Host & Microbe, vol. 21, no. 4, pp. 433–442,
pp. 220–226, 2013. 2017.
[40] E. Pessione, “Lactic acid bacteria contribution to gut microbi- [56] B. Lim, M. Zimmermann, N. A. Barry, and A. L. Goodman,
ota complexity: lights and shadows,” Frontiers in Cellular and “Engineered regulatory systems modulate gene expression
Infection Microbiology, vol. 2, 2012. of human commensals in the gut,” Cell, vol. 169, no. 3,
[41] T. A. Fuchs, U. Abed, C. Goosmann et al., “Novel cell death pp. 547–558.e15, 2017.
program leads to neutrophil extracellular traps,” Journal of [57] W. R. Whitaker, E. S. Shepherd, and J. L. Sonnenburg, “Tun-
Cell Biology, vol. 176, no. 2, pp. 231–241, 2007. able expression tools enable single-cell strain distinction in
[42] T. Wang, J. Fu, X. Xiao et al., “CBP22, a Novel Bacteriocin the gut microbiome,” Cell, vol. 169, no. 3, pp. 538–546.e12,
Isolated from Clostridium butyricum ZJU-F1, Protects 2017.
against LPS-Induced Intestinal Injury through Maintaining [58] M. Barbier, J. Bevere, and F. H. Damron, “In vivo bacterial
the Tight Junction Complex,” Mediators of Inflammation, imaging using bioluminescence,” in Methods In Molecular
vol. 2021, Article ID 8032125, 12 pages, 2021. Biology, pp. 87–97, Springer, (Clifton, N.J), 2018.
[43] D. Begde, S. Bundale, P. Mashitha, J. Rudra, N. Nashikkar, [59] M. E. V. Johansson and G. C. Hansson, “Preservation of
and A. Upadhyay, “Immunomodulatory efficacy of nisin–a mucus in histological sections, immunostaining of mucins
bacterial lantibiotic peptide,” Journal of Peptide Science, in fixed tissue, and localization of bacteria with FISH,” in
vol. 17, no. 6, pp. 438–444, 2011. Methods in Molecular Biology, vol. 842, pp. 229–235, Clifton,
[44] X. Yin, D. Heeney, Y. Srisengfa, B. Golomb, S. Griffey, and N.J, 2012.
M. Marco, “Bacteriocin biosynthesis contributes to the anti- [60] Y. Imai, K. J. Meyer, A. Iinishi et al., “A new antibiotic selec-
inflammatory capacities of probiotic Lactobacillus plan- tively kills gram-negative pathogens,” Nature, vol. 576,
tarum,” Beneficial Microbes, vol. 9, no. 2, pp. 333–344, 2018. no. 7787, pp. 459–464, 2019.
[45] H. N. Bell, R. J. Rebernick, J. Goyert et al., “Reuterin in the [61] W. Wang and X. Chen, “Antibiotics-based fluorescent probes
healthy gut microbiome suppresses colorectal cancer growth for selective labeling of gram-negative and gram-positive bac-
through altering redox balance,” Cancer Cell, vol. 40, no. 2, teria in living microbiotas,” Science China Chemistry, vol. 61,
pp. 185–200.e6, 2022. no. 7, pp. 792–796, 2018.
[46] Z. Norouzi, A. Salimi, R. Halabian, and H. Fahimi, “Nisin, a [62] W. Wang, Y. Zhu, and X. Chen, “Selective imaging of gram-
potent bacteriocin and anti-bacterial peptide, attenuates negative and gram-positive microbiotas in the mouse gut,”
expression of metastatic genes in colorectal cancer cell lines,” Biochemistry, vol. 56, no. 30, pp. 3889–3893, 2017.
Microbial Pathogenesis, vol. 123, pp. 183–189, 2018. [63] F. Faber, L. Tran, M. X. Byndloss et al., “Host-mediated sugar
[47] A. De Giani, F. Bovio, M. Forcella, P. Fusi, G. Sello, and P. Di oxidation promotes post-antibiotic pathogen expansion,”
Gennaro, “Identification of a bacteriocin-like compound Nature, vol. 534, no. 7609, pp. 697–699, 2016.
from Lactobacillus plantarum with antimicrobial activity [64] F. Rivera-Chávez, L. F. Zhang, F. Faber et al., “Depletion of
and effects on normal and cancerogenic human intestinal Butyrate-Producing Clostridia from the Gut Microbiota
cells,” AMB Express, vol. 9, no. 1, 2019. Drives an Aerobic Luminal Expansion of Salmonella,” Cell
[48] P. Baindara, S. Korpole, and V. Grover, “Bacteriocins: per- Host & Microbe, vol. 19, no. 4, pp. 443–454, 2016.
spective for the development of novel anticancer drugs,” [65] W. Wang, L. Lin, Y. Du et al., “Assessing the viability of trans-
Applied Microbiology and Biotechnology, vol. 102, no. 24, planted gut microbiota by sequential tagging with D-amino
pp. 10393–10408, 2018. acid-based metabolic probes,” Nature Communications,
[49] S. Kaur and S. Kaur, “Bacteriocins as potential anticancer vol. 10, no. 1, 2019.
agents,” Frontiers in Pharmacology, vol. 6, 2015. [66] J. Chen, S. Feng, M. Chen et al., “In vivo dynamic monitoring
[50] J. W. Goyert, H. N. Bell, and Y. M. Shah, “PCL22-188: reu- of bacterial infection by NIR-II fluorescence imaging,” Small,
terin in the healthy gut microbiome suppresses colorectal vol. 16, no. 34, article 2002054, 2020.
cancer growth through altering redox balance,” Journal of [67] E. Ferrer-González, J. Fujita, T. Yoshizawa et al., “Structure-
the National Comprehensive Cancer Network, vol. 20, guided design of a fluorescent probe for the visualization
no. 3.5, pp. PCL22–PC188, 2022. of FtsZ in clinically important gram-positive and gram-
[51] E. Meade, M. A. Slattery, and M. Garvey, “Bacteriocins, negative bacterial pathogens,” Scientific Reports, vol. 9,
potent antimicrobial peptides and the fight against multi drug no. 1, 2019.
resistant species: resistance is futile?,” Antibiotics, vol. 9, no. 1, [68] L. Pasquina-Lemonche, J. Burns, R. D. Turner et al., “The
p. 32, 2020. architecture of the gram-positive bacterial cell wall,” Nature,
[52] H. Mathur, D. Field, M. C. Rea, P. D. Cotter, C. Hill, and R. P. vol. 582, no. 7811, pp. 294–297, 2020.
Ross, “Fighting biofilms with lantibiotics and other groups of [69] W. M. Leevy, S. T. Gammon, J. R. Johnson et al., “Noninva-
bacteriocins,” npj Biofilms and Microbiomes, vol. 4, no. 1, p. 9, sive optical imaging of staphylococcus aureus bacterial infec-
2018. tion in living mice using a Bis-dipicolylamine-zinc (II)
Advanced Gut & Microbiome Research 9

affinity group conjugated to a near-infrared fluorophore,” biota of autistic children using an in vitro gut microbiome
Bioconjugate Chemistry, vol. 19, no. 3, pp. 686–692, 2008. model,” Food Research International, vol. 149, article
[70] B.-H. Gan, T. N. Siriwardena, S. Javor, T. Darbre, and 110657, 2021.
J.-L. Reymond, “Fluorescence imaging of bacterial killing [85] M. Marzorati, P. Van den Abbeele, S. Bubeck, T. Bayne,
by antimicrobial peptide dendrimer G3KL,” ACS infec- K. Krishnan, and A. Young, “Treatment with a spore-based
tious diseases, vol. 5, no. 12, pp. 2164–2173, 2019. probiotic containing five strains of Bacillus induced changes
[71] Y. Li, F. Liu, J. Zhang et al., “Efficient killing of multidrug- in the metabolic activity and community composition of the
resistant internalized bacteria by AIEgens in vivo,” Advanced gut microbiota in a SHIME ® model of the human gastroin-
Science, vol. 8, no. 9, article 2001750, 2021. testinal system,” Food Research International, vol. 149, article
[72] F. Hu, G. Qi, D. Mao et al., “Visualization and in situ ablation 110676, 2021.
of intracellular bacterial pathogens through metabolic label- [86] M. Anjum, A. Laitila, A. C. Ouwehand, and S. D. Forssten,
ing,” Angewandte Chemie International Edition, vol. 59, “Current perspectives on gastrointestinal models to assess
no. 24, pp. 9288–9292, 2020. probiotic-pathogen interactions,” Frontiers in Microbiology,
[73] I. Maslov, A. Bogorodskiy, A. Mishin et al., “Efficient non- vol. 13, 2022.
cytotoxic fluorescent staining of halophiles,” Scientific Reports, [87] Y.-H. Chen, W.-H. Tsai, H.-Y. Wu et al., “Probiotic Lactoba-
vol. 8, no. 1, 2018. cillus spp. act against Helicobacter pylori-induced inflamma-
[74] K. E. Bullok, S. T. Gammon, S. Violini et al., “Permeation tion,” Journal of Clinical Medicine, vol. 8, no. 1, p. 90, 2019.
peptide conjugates for in vivo molecular imaging applica- [88] M. A. Engevik, W. Ruan, M. Esparza et al., “Immunomodula-
tions,” Molecular Imaging, vol. 5, no. 1, pp. 1–15, 2006. tion of dendritic cells by Lactobacillus reuteri surface compo-
[75] N. Nekhotiaeva, A. Elmquist, G. K. Rajarao, M. Hallbrink, nents and metabolites,” Physiological Reports, vol. 9, no. 2,
U. Langel, and L. Good, “Cell entry and antimicrobial prop- article e14719, 2021.
erties of eukaryotic cell-penetrating peptides,” The FASEB [89] V. De Gregorio, C. Sgambato, F. Urciuolo, R. Vecchione,
Journal, vol. 18, no. 2, pp. 1–15, 2004. P. A. Netti, and G. Imparato, “Immunoresponsive microbi-
[76] P. Abishad, J. Vergis, V. Unni et al., “Green synthesized silver ota-gut-on-chip reproduces barrier dysfunction, stromal
nanoparticles using Lactobacillus acidophilus as an antioxi- reshaping and probiotics translocation under inflammation,”
dant, antimicrobial, and antibiofilm agent against multi- Biomaterials, vol. 286, article 121573, 2022.
drug resistant enteroaggregative Escherichia coli,” Probiotics [90] C. Guan, Y. Yang, D. Tian et al., “Evaluation of an Ussing
and Antimicrobial Proteins, vol. 14, no. 5, pp. 904–914, 2022. chamber system equipped with rat intestinal tissues to predict
[77] L. Ruiz, A. B. Flórez, B. Sánchez et al., “Bifidobacterium intestinal absorption and metabolism in humans,” European
longum subsp. infantis CECT7210 (B. infantis IM-1®) dis- Journal of Drug Metabolism and Pharmacokinetics, vol. 47,
plays in vitro activity against some intestinal pathogens,” no. 5, pp. 639–652, 2022.
Nutrients, vol. 12, no. 11, p. 3259, 2020. [91] A. Haque, F. Bowe, R. Fitzhenry et al., “Early interactions of
[78] D. Cizeikiene and J. Jagelaviciute, “Investigation of antibacte- Salmonella enterica serovar Typhimurium with human small
rial activity and probiotic properties of strains belonging to intestinal epithelial explants,” Gut, vol. 53, no. 10, pp. 1424–
Lactobacillus and Bifidobacterium genera for their potential 1430, 2004.
application in functional food and feed products,” Probiotics [92] U. Marx, E. Accastelli, R. David et al., “An individual patient’s
and Antimicrobial Proteins, vol. 13, no. 5, pp. 1387–1403, “body” on chips—how organismoid theory can translate into
2021. your personal precision therapy approach,” Frontiers in Med-
[79] Y. Wei, F. Yang, Q. Wu et al., “Protective effects of bifidobac- icine, vol. 8, 2021.
terial strains against toxigenic Clostridium difficile,” Frontiers [93] C. Scalfaro, A. Iacobino, C. Nardis, and G. Franciosa, “Galle-
in Microbiology, vol. 9, 2018. ria mellonella as an in vivo model for assessing the protective
[80] D. Ghadimi, A. Nielsen, M. F. Hassan et al., “Modulation of activity of probiotics against gastrointestinal bacterial patho-
proinflammatory bacteria-and lipid-coupled intracellular sig- gens,” FEMS Microbiology Letters, vol. 364, no. 7, 2017.
naling pathways in a Transwell triple co-culture model by [94] Z. Zhao, S. Xu, W. Zhang, D. Wu, and G. Yang, “Probiotic
commensal Bifidobacterium animalis R101-8,” Anti-Inflam- Escherichia coli NISSLE 1917 for Inflammatory Bowel Dis-
matory & Anti-Allergy Agents in Medicinal Chemistry, ease Applications,” Food & Function, vol. 13, no. 11,
vol. 20, no. 2, pp. 161–181, 2021. pp. 5914–5924, 2022.
[81] J. C. Arciero, G. B. Ermentrout, J. S. Upperman, Y. Vodovotz, [95] T. M. Dronkers, A. C. Ouwehand, and G. T. Rijkers, “Global
and J. E. Rubin, “Using a mathematical model to analyze the analysis of clinical trials with probiotics,” Heliyon, vol. 6,
role of probiotics and inflammation in necrotizing enteroco- no. 7, article e04467, 2020.
litis,” PLoS One, vol. 5, no. 4, article e10066, 2010. [96] L. Beaulieu, Production, Purification et Caracterisation de la
[82] Y.-M. Choi, Y. Q. Lee, H.-S. Song, and D.-Y. Lee, “Genome Pediocine PA-1 Naturelle et de ses Formes Recombinantes:
scale metabolic models and analysis for evaluating probiotic Contribution a la Mise en Evidence d'une Nouvelle Activite
potentials,” Biochemical Society Transactions, vol. 48, no. 4, Biologique (French and English text), [Ph.D. thesis], Univer-
pp. 1309–1321, 2020. site Laval, Canada, 2004.
[83] A. Gościniak, P. Eder, J. Walkowiak, and J. Cielecka-Piontek, [97] H. Sareh Sadat, H. Bahareh, F. Ebrahim, and G. Hossein,
“Artificial gastrointestinal models for nutraceuticals “Increased expression of caspase genes in colorectal cancer
research—achievements and challenges: a practical review,” cell line by nisin,” Archives of Clinical Infectious Diseases,
Nutrients, vol. 14, no. 13, p. 2560, 2022. vol. 15, no. 2, 2020.
[84] A. L. R. F. Duque, F. M. Demarqui, M. M. Santoni et al., [98] A. Shiva, G. Marzieh, and H. Hamideh Mahmoodzadeh, “The
“Effect of probiotic, prebiotic, and synbiotic on the gut micro- apoptotic impact of nisin as a potent bacteriocin on the colon
10 Advanced Gut & Microbiome Research

cancer cells,” Microbial Pathogenesis, vol. 111, pp. 193–197,

2017.
[99] S. Maher and S. McClean, “Investigation of the cytotoxicity of
eukaryotic and prokaryotic antimicrobial peptides in intesti-
nal epithelial cells in vitro,” Biochemical Pharmacology,
vol. 71, no. 9, pp. 1289–1298, 2006.
[100] L. M. John, “Managing potentially resectable metastatic colon
cancer,” Gastrointestinal Cancer Research: GCR, vol. 2,
Supplement 4, pp. S23–S26, 2008.
[101] N. J. Roberts, L. Zhang, F. Janku et al., “Intratumoral injection
of Clostridium novyi-NT spores induces antitumor responses,”
Science Translational Medicine, vol. 6, no. 249, article
249ra111, 2014.
[102] C. M. Guinane, E. M. Lawton, P. M. O'Connor et al., “The
bacteriocin bactofencin A subtly modulates gut microbial
populations,” Anaerobe, vol. 40, pp. 41–49, 2016.
[103] F. Taghizad, H. R. Kazerani, H. Dehghani, A. Asoodeh, and
D. Yaghubi, “A novel approach towards obesity: the use of a
bacterial product, gassericin A, in 3T3-L1 cells,” Obesity
Research & Clinical Practice, vol. 15, no. 5, pp. 499–505, 2021.