Application of Probiotics and Their Potential Health Benefits

Journal Name	Impact Factor	CiteScore	Launched Year	First Decision (median)	APC
Applied Microbiology applmicrobiol	-	-	2021	14.3 Days	CHF 1000
Biology biology	3.6	5.7	2012	16.4 Days	CHF 2700
Biomolecules biomolecules	4.8	9.4	2011	18.4 Days	CHF 2700
International Journal of Molecular Sciences ijms	4.9	8.1	2000	16.8 Days	CHF 2900
Microorganisms microorganisms	4.1	7.4	2013	11.7 Days	CHF 2700

4 pages, 183 KiB

Open AccessEditorial

Applications of Probiotics and Their Potential Health Benefits

by Virginia Fuochi and Pio Maria Furneri

Int. J. Mol. Sci. 2023, 24(21), 15915; https://doi.org/10.3390/ijms242115915 - 2 Nov 2023

Cited by 3 | Viewed by 4168

Probiotics have garnered significant attention in recent years due to their potential health benefits and their role in promoting a balanced gut microbiome [...] Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

20 pages, 4562 KiB

Open AccessArticle

Beneficial Effects of Bacillus amyloliquefaciens D1 Soy Milk Supplementation on Serum Biochemical Indexes and Intestinal Health of Bearded Chickens

by Liyu Du, Weizhe Chen, Jie Wang, Lingzhu Huang, Qikai Zheng, Junjie Chen, Linhao Wang, Changyu Cai, Xiangbin Zhang, Li Wang, Qingping Zhong, Wujie Zhong, Xiang Fang and Zhenlin Liao

Microorganisms 2023, 11(7), 1660; https://doi.org/10.3390/microorganisms11071660 - 26 Jun 2023

Cited by 5 | Viewed by 1997

Abstract

This study investigated the effects of dietary supplementation with Bacillus amyloliquefaciens D1 (B. amyloliquefaciens D1) on growth performance, serum anti-inflammatory cytokines, and intestinal microbiota composition and diversity in bearded chickens. To investigate the effects of Bacillus amyloliquefaciensa and fermented soy milk, 7-day-old [...] Read more.

This study investigated the effects of dietary supplementation with Bacillus amyloliquefaciens D1 (B. amyloliquefaciens D1) on growth performance, serum anti-inflammatory cytokines, and intestinal microbiota composition and diversity in bearded chickens. To investigate the effects of Bacillus amyloliquefaciensa and fermented soy milk, 7-day-old broilers were orally fed different doses of Bacillus amyloliquefaciens D1 fermented soy milk for 35 days, with the unfermented soy milk group as the Placebo group. This study found that B. amyloliquefaciens D1 fermented soy milk improved the intestinal microbiota of broilers, significantly increasing the abundance of beneficial bacteria and decreasing the abundance of harmful bacteria in the gut. B. amyloliquefaciens D1 fermented soy milk also significantly reduced the serum lipopolysaccharide (LPS) content. The body weight and daily weight gain of broilers were increased. In conclusion, the results of this study are promising and indicate that supplementing the diets of bearded chickens with B. amyloliquefaciens D1 fermented soy milk has many beneficial effects in terms of maintaining intestinal microbiota balance and reducing inflammation in chickens. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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17 pages, 3416 KiB

Open AccessArticle

Functional Characterization of Fermented Beverages Based on Soy Milk and Sea Buckthorn Powder

by Nicoleta-Maricica Maftei, Alina-Viorica Iancu, Alina Mihaela Elisei, Tudor Vladimir Gurau, Ana Yndira Ramos-Villarroel and Elena Lacramioara Lisa

Microorganisms 2023, 11(6), 1493; https://doi.org/10.3390/microorganisms11061493 - 4 Jun 2023

Cited by 13 | Viewed by 2465

Abstract

Limitations of dairy products, such as lactose intolerance, problems related to a high cholesterol intake in diet, malabsorption, and the requirement for cold storage facilities, as well as an increasing demand for new foods and tastes, have initiated a trend in the development [...] Read more.

Limitations of dairy products, such as lactose intolerance, problems related to a high cholesterol intake in diet, malabsorption, and the requirement for cold storage facilities, as well as an increasing demand for new foods and tastes, have initiated a trend in the development of non-dairy probiotic products. The possibility of producing beverages based on soy milk, sea buckthorn powder, and fermented by Bifidobacterium bifidus (Bb-12^®, Bb) strain at different temperatures (30 °C and 37 °C) was examined. Strain viability, pH, and titratable acidity were measured during the fermentation period while the viability, pH, titratable acidity, and water holding capacity were determined during the storage time at 4 °C ± 1 °C within 14 days. Additionally, the survival and stability of Bb-12^®, inoculated into a functional beverage when exposed to simulated gastrointestinal tract conditions, were assessed. The results obtained in this study revealed that the content of potent bioactive compounds in fermented soy milk and sea buckthorn powder depends on the processing conditions, the bacteria used in the fermentation step, and storage time. Full article

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25 pages, 1560 KiB

Open AccessArticle

Sow-Offspring Diets Supplemented with Probiotics and Synbiotics Are Associated with Offspring’s Growth Performance and Meat Quality

by Qian Zhu, Md. Abul Kalam Azad, Haibo Dong, Chenjian Li, Ruixuan Li, Yating Cheng, Yang Liu, Yulong Yin and Xiangfeng Kong

Int. J. Mol. Sci. 2023, 24(8), 7668; https://doi.org/10.3390/ijms24087668 - 21 Apr 2023

Cited by 5 | Viewed by 2826

Abstract

Probiotics and synbiotics supplementation have been shown to play potential roles in animal production. The present study aimed to evaluate the effects of dietary probiotics and synbiotics supplementation to sows during gestation and lactation and to offspring pigs (sow-offspring) on offspring pigs’ growth [...] Read more.

Probiotics and synbiotics supplementation have been shown to play potential roles in animal production. The present study aimed to evaluate the effects of dietary probiotics and synbiotics supplementation to sows during gestation and lactation and to offspring pigs (sow-offspring) on offspring pigs’ growth performance and meat quality. Sixty-four healthy Bama mini-pigs were selected and randomly allocated into four groups after mating: the control, antibiotics, probiotics, and synbiotics groups. After weaning, two offspring pigs per litter were selected, and four offspring pigs from two litters were merged into one pen. The offspring pigs were fed a basal diet and the same feed additive according to their corresponding sows, representing the control group (Con group), sow-offspring antibiotics group (S-OA group), sow-offspring probiotics group (S-OP group), and sow-offspring synbiotics group (S-OS group). Eight pigs per group were euthanized and sampled at 65, 95, and 125 d old for further analyses. Our findings showed that probiotics supplementation in sow-offspring diets promoted growth and feed intake of offspring pigs during 95–125 d old. Moreover, sow-offspring diets supplemented with probiotics and synbiotics altered meat quality (meat color, pH_45min, pH_24h, drip loss, cooking yield, and shear force), plasma UN and AMM levels, and gene expressions associated with muscle-fiber types (MyHCI, MyHCIIa, MyHCIIx, and MyHCIIb) and muscle growth and development (Myf5, Myf6, MyoD, and MyoG). This study provides a theoretical basis for the maternal-offspring integration regulation of meat quality by dietary probiotics and synbiotics supplementation. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Effects of probiotics and synbiotics supplementation in sow-offspring diets on mRNA expressions of myosin heavy chain (MyHC) isoforms and myogenic regulatory factors (MRFs) in the skeletal muscle of offspring pigs at 65, 95, and 125 d old. (A) and (E) are mRNA expressions of MyHC isoforms in the longissimus doris muscle (LDM) and psoas major muscle (PMM), respectively. (B–D) and (F−H) are mRNA expressions of MRFs in the LDM and PMM, respectively. a–c Different letters mean significant differences (p < 0.05). MyHCI, myosin heavy chain I; MyHCIIa, myosin heavy chain IIa; MyHCIIb, myosin heavy chain IIb; MyHCIIx, myosin heavy chain IIx; MyoD, myogenic differentiation factor; MyoG, myogenin; Myf5, myogenic factor 5; Myf6, myogenic factor 6; IGF1, insulin-like growth factor 1; MAFbx, muscle atrophy Fbox-1 protein; MSTN, myostatin. Con group: sow and offspring pigs fed with a basal diet; S-OA group: sow and offspring pigs fed with antibiotics; S-OP group: sow and offspring pigs fed with probiotics; S-OS group: sow and offspring pigs fed with synbiotics. The replicates per group at 65 d old were 8. The replicates of the Con, S-OA, S-OP, and S-OS groups at 95 d old were 8, 8, 8, and 7, respectively. The replicates of the Con, S-OA, S-OP, and S-OS groups at 125 d old were 8, 5, 6, and 6, respectively. Full article ">Figure 2
Schematic presentation of the experimental design. Full article ">

24 pages, 2606 KiB

Open AccessReview

Can Probiotics, Particularly Limosilactobacillus fermentum UCO-979C and Lacticaseibacillus rhamnosus UCO-25A, Be Preventive Alternatives against SARS-CoV-2?

by Héctor Valdebenito-Navarrete, Victor Fuentes-Barrera, Carlos T. Smith, Alexis Salas-Burgos, Felipe A. Zuniga, Leonardo A. Gomez and Apolinaria García-Cancino

Biology 2023, 12(3), 384; https://doi.org/10.3390/biology12030384 - 28 Feb 2023

Cited by 5 | Viewed by 3475

Abstract

COVID-19, an infection produced by the SARS-CoV-2 virus in humans, has rapidly spread to become a high-mortality pandemic. SARS-CoV-2 is a single-stranded RNA virus characterized by infecting epithelial cells of the intestine and lungs, binding to the ACE2 receptor present on epithelial cells. [...] Read more.

COVID-19, an infection produced by the SARS-CoV-2 virus in humans, has rapidly spread to become a high-mortality pandemic. SARS-CoV-2 is a single-stranded RNA virus characterized by infecting epithelial cells of the intestine and lungs, binding to the ACE2 receptor present on epithelial cells. COVID-19 treatment is based on antivirals and antibiotics against symptomatology in addition to a successful preventive strategy based on vaccination. At this point, several variants of the virus have emerged, altering the effectiveness of treatments and thereby attracting attention to several alternative therapies, including immunobiotics, to cope with the problem. This review, based on articles, patents, and an in silico analysis, aims to address our present knowledge of the COVID-19 disease, its symptomatology, and the possible beneficial effects for patients if probiotics with the characteristics of immunobiotics are used to confront this disease. Moreover, two probiotic strains, L. fermentum UCO-979C and L. rhamnosus UCO-25A, with different effects demonstrated at our laboratory, are emphasized. The point of view of this review highlights the possible benefits of probiotics, particularly those associated with immunomodulation as well as the production of secondary metabolites, and their potential targets during SARS-CoV-2 infection. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Intestinal eubiotic and SARS-CoV-2-induced intestinal dysbiosis. (A) In a general intestinal environment in eubiotic conditions, tryptophan can enter into the intestinal tract’s epithelial cells, generating the production of antimicrobial peptides that will regulate the intestinal microbiota. In addition, ACE2 can convert Ang II into Ang (1-7), which, after binding to its receptor, Mas, blocks intestinal glucose transport. (B) The various proinflammatory mechanisms caused during SARS-CoV-2 infection generate an intestinal dysbiosis condition. Under this condition, SARS-CoV-2 can block the B°AT1 receptor, preventing both the passage of tryptophan into the epithelial cells and the production of antimicrobial peptides. SARS-CoV-2 can also disrupt the tight junctions of the cells, producing leaky gut syndrome and inflammation. Moreover, SARS-CoV-2 binds to the ACE2 receptor, preventing the formation of Ang (1-7) and favoring the activity of the SGLT1 as well as GLUT2 transporters, increasing the intestinal glucose levels. Full article ">Figure 2
Mechanisms of action of probiotics. Several mechanisms are associated with the antagonistic effect of probiotics against intestinal pathogens, including the following: (a). Enhancement of the epithelial barrier function. Probiotic bacteria can maintain the barrier’s integrity, favoring its adhesion to the intestinal mucosa and maintaining the epithelial junctional complex, preventing the entry of pathogens or metabolites that may cause inflammatory responses. (b). Competition for nutrients and prebiotics. Probiotic bacteria are capable of competing for nutrients that are necessary for the survival of pathogens. (c). Bioconversion. Commensal bacteria can transform organic compounds into bioactive metabolites to generate a better environment. (d). Direct antagonism. Probiotic bacteria can produce bacteriocins and other compounds that can attack pathogens. (e). Production of growth inhibitors. Probiotic bacteria can produce compounds that inhibit the growth of some pathogenic bacteria. (f). Competitive exclusion. The mechanism by which certain bacteria are better competitors than pathogens for receptors. (g). Inflammation reduction. Commensal bacteria are capable of inducing an anti-inflammatory response, causing the inhibition of Th1, Th2, and Th17. (h). Immune system modulation. Bacteria can interact with dendritic cells, causing an immunomodulating effect and inducing B and T cell responses. Full article ">Figure 3
The intestine–lung axis. The regulation of lung immunity by the gastrointestinal microbiota is based on three basic alternatives. The first is the production of antibodies (immunoglobulin A) by activating B cells during the stimulation of the immune system by commensal intestinal bacteria. The second is the activation of T cells, stimulated by the dendritic cells interacting with commensal intestinal bacteria. The third is the generation of bacterial metabolites by the intestinal microbiota. All three can pass by the lymphatic system and reach the lungs, where they can generate responses against respiratory pathogens. Full article ">Figure 4
Mechanism of action of probiotics against SARS-CoV-2 infection. SARS-CoV-2 binds to ACE2 respiratory and intestinal cell receptors, producing dysbiosis of the intestinal microbiota. Probiotics can induce innate-immunity-activating cells, such as NK cells, polymorphonuclear (PMN) leukocytes, and macrophages, by means of the activity of cytokines, triggering responses with an immunomodulatory effect and also being capable of producing regulated inflammatory reactions through the action of cytokines. Full article ">

14 pages, 2384 KiB

Open AccessArticle

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development

by Ke Ma, Wei Chen, Xiao-Qi Lin, Zhen-Zhen Liu, Tao Wang, Jia-Bao Zhang, Jian-Gang Zhang, Cheng-Kai Zhou, Yu Gao, Chong-Tao Du and Yong-Jun Yang

Int. J. Mol. Sci. 2023, 24(3), 3045; https://doi.org/10.3390/ijms24033045 - 3 Feb 2023

Cited by 11 | Viewed by 3562

Abstract

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and [...] Read more.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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14 pages, 1927 KiB

Open AccessArticle

Stimulation of Heme-Dependent Catalase Enhanced the Cytoprotective Effect of Lactobacillus plantarum against Oxidative Stress

by Xingfang Tian, Xiaoce Zhu, Meng Wang, Tingting Guo and Jian Kong

Appl. Microbiol. 2023, 3(1), 131-144; https://doi.org/10.3390/applmicrobiol3010011 - 23 Jan 2023

Cited by 2 | Viewed by 2416

Abstract

Lactobacillus plantarum is a catalase-negative species and distributes in human intestinal tracts. However, the cytoprotective effects of the catalase-activated L. plantarum strain have yet to be exploited against reactive oxygen species (ROS). Here, a catalase-activated L. plantarum CGMCC 6888 (CatA⁺) was obtained [...] Read more.

Lactobacillus plantarum is a catalase-negative species and distributes in human intestinal tracts. However, the cytoprotective effects of the catalase-activated L. plantarum strain have yet to be exploited against reactive oxygen species (ROS). Here, a catalase-activated L. plantarum CGMCC 6888 (CatA⁺) was obtained using exogenous added heme. The scavenging free radical abilities of this strain were obviously increased. Moreover, the activated catalase A in L. plantarum CGMCC 6888 endowed the intestinal epithelium NCM460 with lower ROS content after degrading H₂O₂. In addition, the transcription levels of Nrf2 and Nrf2-related antioxidant enzyme genes (HO-1, GCLC, NQO-1 and TXNRD1) and tight junction protein genes (ZO-1, OCLN, and JAM-1) were upregulated significantly when co-incubated with CGMCC 6888/CatA⁺. This work confirmed that the catalase A conferred L. plantarum with the strong protection effects in the intestinal epithelial cells against ROS. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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19 pages, 7728 KiB

Open AccessArticle

Supplementation of Weizmannia coagulans BC2000 and Ellagic Acid Inhibits High-Fat-Induced Hypercholesterolemia by Promoting Liver Primary Bile Acid Biosynthesis and Intestinal Cholesterol Excretion in Mice

by Long Jin, Hongyang Dang, Jinyong Wu, Lixia Yuan, Xiangsong Chen and Jianming Yao

Microorganisms 2023, 11(2), 264; https://doi.org/10.3390/microorganisms11020264 - 19 Jan 2023

Cited by 6 | Viewed by 2642

Abstract

The probiotic Weizmannia coagulans (W. coagulans) BC2000 can increase the abundance of intestinal transforming ellagic acid (EA) bacteria and inhibit metabolic disorders caused by hyperlipidemia by activating liver autophagy. This study aimed to investigate the inhibitory effects of W. coagulans BC2000 [...] Read more.

The probiotic Weizmannia coagulans (W. coagulans) BC2000 can increase the abundance of intestinal transforming ellagic acid (EA) bacteria and inhibit metabolic disorders caused by hyperlipidemia by activating liver autophagy. This study aimed to investigate the inhibitory effects of W. coagulans BC2000 and EA on hyperlipidemia-induced cholesterol metabolism disorders. C57BL/6J mice (n = 10 in each group) were fed a low-fat diet, high-fat diet (HFD), HFD supplemented with EA, HFD supplemented with EA and W. coagulans BC77, HFD supplemented with EA, and W. coagulans BC2000. EA and W. coagulans BC2000 supplementation prevented HFD-induced hypercholesterolemia and promoted fecal cholesterol excretion. Transcriptome analysis showed that primary bile acid biosynthesis in the liver was significantly activated by EA and W. coagulans BC2000 treatments. EA and W. coagulans BC2000 treatment also significantly increased the intestinal Eggerthellaceae abundance and the liver EA metabolites, iso-urolithin A, Urolithin A, and Urolithin B. Therefore, W. coagulans BC2000 supplementation promoted the intestinal transformation of EA, which led to the upregulation of liver bile synthesis, thus preventing hypercholesterolemia. Full article

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36 pages, 2313 KiB

Open AccessReview

Journey of the Probiotic Bacteria: Survival of the Fittest

by Allyson Andrade Mendonça, Walter de Paula Pinto-Neto, Giselle Alves da Paixão, Dayane da Silva Santos, Marcos Antonio De Morais, Jr. and Rafael Barros De Souza

Microorganisms 2023, 11(1), 95; https://doi.org/10.3390/microorganisms11010095 - 30 Dec 2022

Cited by 28 | Viewed by 9990

Abstract

This review aims to bring a more general view of the technological and biological challenges regarding production and use of probiotic bacteria in promoting human health. After a brief description of the current concepts, the challenges for the production at an industrial level [...] Read more.

This review aims to bring a more general view of the technological and biological challenges regarding production and use of probiotic bacteria in promoting human health. After a brief description of the current concepts, the challenges for the production at an industrial level are presented from the physiology of the central metabolism to the ability to face the main forms of stress in the industrial process. Once produced, these cells are processed to be commercialized in suspension or dried forms or added to food matrices. At this stage, the maintenance of cell viability and vitality is of paramount for the quality of the product. Powder products requires the development of strategies that ensure the integrity of components and cellular functions that allow complete recovery of cells at the time of consumption. Finally, once consumed, probiotic cells must face a very powerful set of physicochemical mechanisms within the body, which include enzymes, antibacterial molecules and sudden changes in pH. Understanding the action of these agents and the induction of cellular tolerance mechanisms is fundamental for the selection of increasingly efficient strains in order to survive from production to colonization of the intestinal tract and to promote the desired health benefits. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Journey of probiotic products from their production in the industry to their arrival in the intestine, where they will perform their beneficial functions for consumers. In this path, the main challenges and activities of the cells are highlighted. Full article ">Figure 2
Overview of consumption of different types of monosaccharide sugars (glucose, fructose, galactose, mannose, arabinose and xylose), disaccharides (sucrose, lactose, maltose, cellobiose and xylobiose), trisaccharides (raffinose and panose) and oligosaccharides (fructooligosaccharide, galactooligosaccharide and xylooligosaccharide) and their integration into the central carbon metabolism of probiotic bacteria. Sugars composed of more than one unit must be hydrolysed during passage through transporters or by internal glycohydrolases to release the constituent monosaccharides. Hexoses are converted to glucose 6-phosphate (GLU-6P) or fructose 6-phosphate (FRU-6P). The exception is galactose, which initially must be metabolized by the Leloir pathway. Next, GLU-6P and FRU-6P are metabolized via the glycolytic pathway. GLU-6P can also be metabolized via the pentose phosphate pathway (PPP) to subsequently return to the glycolytic pathway. Pentoses, in turn, are converted directly by PPP. The intermediate xylulose 5-phosphate (XYLU-5P) from GLU-6P or pentoses can be broken down into a molecule of glyceraldehyde 3-phosphate (GA3P) and a molecule of acetyl phosphate (ACE-P) by the enzyme phosphoketolase (PK). Full article ">Figure 3
Types of fermentative metabolism displayed by probiotic bacteria. Exclusive homofermenter bacteria utilize glucose or other hexoses exclusively via the glycolytic pathway (a). Obligate heterofermenters utilize the PPP pathway and phosphoketolase activity (PPP-PK) to metabolize hexoses and pentoses (b). Facultative heterofermenters use the glycolytic pathway to ferment hexoses and the PPP-PK pathway to ferment pentoses (c). Full article ">Figure 4
Action of lysozyme on the structure of the peptidoglycan component of the cell wall of probiotic bacteria. In sensitive strains, the enzyme hydrolyses the bond between N-acetylglucosamine (NAG) and N-Acetylmuramic acid (NAM). In resistant strains, NAM residues undergo O-acetylation at the C-6 position. This makes the NAG-NAM bond inaccessible to lysozyme. Full article ">Figure 5
Overview of the main mechanisms of acid stress tolerance in lactic acid bacteria involving amino acid catabolism, expression of stress response metabolism genes by sigma alternative transcription factors activated by histidine kinase and activation of the ATP-dependent proton extrusion pump. Full article ">Figure 6
Overview of the main mechanisms of tolerance to intestinal tract stress conditions in lactic acid bacteria from the decomposition of reactive oxygen species (ROS), hydrolysis of bile salts and their excretion by extrusion pumps, antiport ion transport pumps and activation alkaline shock response proteins. Full article ">

17 pages, 1786 KiB

Open AccessArticle

Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs

by Monika Moravkova, Iveta Kostovova, Katerina Kavanova, Radko Pechar, Stanislav Stanek, Ales Brychta, Michal Zeman and Tereza Kubasova

Microorganisms 2023, 11(1), 103; https://doi.org/10.3390/microorganisms11010103 - 30 Dec 2022

Cited by 6 | Viewed by 2244

Abstract

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our [...] Read more.

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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The phylogenetic tree of a tetW gene, showing the relationship between the tetW genes from L. amylovorus (received from our study and NCBI) and the tetW sequences of selected bacterial species showing the highest similarity to L. amylovorus tetW genes (based on the blastn analysis). The evolutionary history was inferred using the Maximum Likelihood method and the Tamura 3-parameter model [<a href="#B36-microorganisms-11-00103" class="html-bibr">36</a>]. Bootstrap values (1000 replicates) were applied and the percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. There were a total of 1932 positions in the final dataset. The phylogenetic tree was rooted with the tetW from Bifidobacterium longum subsp. suillum JCM 19995 as an outgroup. Evolutionary analyses were conducted in MEGA X [<a href="#B34-microorganisms-11-00103" class="html-bibr">34</a>]. Full article ">Figure 2
Organization of CDS in the contigs harboring tetW and xerC/D in L. amylovorus strains and comparison with plasmid pPMRA301 and plasmid p2 from L. amylovorus PMRA3 and GLR1118, respectively. Yellow arrow—CDS associated with mobility (e.g., IS—transposase, tr—putative transposase, int—putative integrase), red arrow—tetW gene, teal arrow—CDS with COG/PROKKA annotation, gray arrow—hypothetical protein, orange—XerC/D site-specific recombinase, green rectangle—tetW regulatory protein (trp), light blue—unknown misc. feature. The gray zones between sequences represent blastn sequence identity. *Plasmid p2 from GLR1118 shown only CDS identical to contigs bearing tetW resistance. Full article ">Figure 3
Organization of CDS in the contigs harboring tetW and site-specific recombinase spoIVCA or tetW and xre (DNA-binding transcriptional regulator) in L. amylovorus strains. Yellow arrow—CDS associated with mobility (e.g., IS—transposase), red arrow—tetW gene, teal arrow—CDS with functional annotation, gray arrow—hypothetical protein, orange—SpoIVCA site-specific recombinase, green rectangle—tetW regulatory protein (trp), blue arrow—CDS identified in other bacterial spp. (e.g., Treponema succinifaciens DSM 2489 or Victivallales bacterium CCUG 44730). The gray zones between sequences represent blastn sequence identity (generated by EasyFig). Full article ">Figure 4
Organization of CDS in the contigs harboring ermB gene. (A) contigs bearing omega transcriptional repressor (omtr) near ermB. (B) CDS pattern with mobilization protein (mp) near the ermB gene. (A) and (B): dark blue arrow—23S rRNA methyl transferase (rmt) and rRNA adenine methyltransferase gene (ramt), red arrow—ermB gene, teal arrow—CDS with functional annotation, gray arrow—hypothetical protein, pink arrow—omega transcriptional repressor (omtr), light blue rectangle—palindromatic sequences, yellow arrow—CDS associated with mobility (IS—transposase). The gray zones between sequences represent blastn sequence identity. Full article ">

17 pages, 3208 KiB

Open AccessArticle

Transcriptomic Analysis Revealed Antimicrobial Mechanisms of Lactobacillus rhamnosus SCB0119 against Escherichia coli and Staphylococcus aureus

by Huan Peng, Gang Zhou, Xi-Miao Yang, Guo-Jun Chen, Hai-Bin Chen, Zhen-Lin Liao, Qing-Ping Zhong, Li Wang, Xiang Fang and Jie Wang

Int. J. Mol. Sci. 2022, 23(23), 15159; https://doi.org/10.3390/ijms232315159 - 2 Dec 2022

Cited by 7 | Viewed by 3284

Abstract

Lactic acid bacteria were reported as a promising alternative to antibiotics against pathogens. Among them, Lactobacillus rhamnosus could be used as probiotics and inhibit several pathogens, but its antibacterial mechanisms are still less known. Here, L. rhamnosus SCB0119 isolated from fermented pickles could [...] Read more.

Lactic acid bacteria were reported as a promising alternative to antibiotics against pathogens. Among them, Lactobacillus rhamnosus could be used as probiotics and inhibit several pathogens, but its antibacterial mechanisms are still less known. Here, L. rhamnosus SCB0119 isolated from fermented pickles could inhibit bacterial growth or even cause cell death in Escherichia coli ATCC25922 and Staphylococcus aureus ATCC6538, which was mainly attributed to the cell-free culture supernatant (CFS). Moreover, CFS induced the accumulation of reactive oxygen species and destroyed the structure of the cell wall and membrane, including the deformation in cell shape and cell wall, the impairment of the integrity of the cell wall and inner membrane, and the increases in outer membrane permeability, the membrane potential, and pH gradient in E. coli and S. aureus. Furthermore, the transcriptomic analysis demonstrated that CFS altered the transcripts of several genes involved in fatty acid degradation, ion transport, and the biosynthesis of amino acids in E. coli, and fatty acid degradation, protein synthesis, DNA replication, and ATP hydrolysis in S. aureus, which are important for bacterial survival and growth. In conclusion, L. rhamnosus SCB0119 and its CFS could be used as a biocontrol agent against E. coli and S. aureus. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Figure 1
Antimicrobial activity of L. rhamnosus SCB0119 against E. coli ATCC25922 and S. aureus ATCC6538. (A) The mean diameters of inhibition zones after E. coli ATCC25922 and S. aureus ATCC6538 were incubated with L. rhamnosus SCB0119 culture solution and CFS for 12 h, respectively. (B,C) Effects of CFS on the growth of E. coli ATCC25922 and S. aureus ATCC6538, respectively. Error bars: SD of the mean from three repeated assays. Full article ">Figure 2
Effects of CFS from L. rhamnosus SCB0119 on the production of ATP (A,B) and ROS (C) in E. coli ATCC25922 (A,C) and S. aureus ATCC6538 (B,C). ROS production was detected using DCFH-DA staining. Different lowercase letters indicate significant differences among the treatments (p < 0.05). Error bars: SD of the mean from three repeated assays. Full article ">Figure 3
SEM and TEM images after E. coli ATCC25922 and S. aureus ATCC6538 cells were treated with 1/2 MIC of CFS from L. rhamnosus SCB0119. Full article ">Figure 4
Effects of CFS from L. rhamnosus SCB0119 on AKP contents (A), outer membrane permeability (B), inner membrane integrity (C), membrane potential (D) and pH gradients (E) in E. coli ATCC25922 and S. aureus ATCC6538. Changes in the outer membrane permeability, inner membrane integrity, membrane potential, and pH gradients were assayed using NPN, PI, DiBAC4(3), and BCECF-AM staining, respectively. Different lowercase letters indicate significant differences among the treatments (p < 0.05). Error bars: SD of the mean from three repeated assays. Full article ">Figure 5
Fold change of selected genes by qRT-PCR after bacterial cells were treated with CFS from L. rhamnosus SCB0119 in E. coli ATCC25922 (A) and S. aureus ATCC6538 (B). The bacterial 16S rRNA was used as an internal standard. The red line at value 1 represented the expression of chosen genes in the control groups. Error bars: SD of the mean from three repeated assays. Full article ">Figure 6
The GO enrichment scatter plot (A) and KEGG enrichment scatter plot (B) of differentially expressed genes treated with CFS from L. rhamnosus SCB0119 in E. coli ATCC25922. Full article ">Figure 7
The GO enrichment scatter plot (A) and KEGG enrichment scatter plot (B) of differentially expressed genes treated with CFS from L. rhamnosus SCB0119 in S. aureus ATCC6538. Full article ">

16 pages, 3311 KiB

Open AccessArticle

Bifidobacterium longum, Lactobacillus plantarum and Pediococcus acidilactici Reversed ETEC-Inducing Intestinal Inflammation in Mice

by Wentao Li, Lixia Kai, Zipeng Jiang, Huan He, Mingzhi Yang, Weifa Su, Yizhen Wang, Mingliang Jin and Zeqing Lu

Microorganisms 2022, 10(12), 2350; https://doi.org/10.3390/microorganisms10122350 - 28 Nov 2022

Cited by 10 | Viewed by 3226

Abstract

Microecological preparation could relieve Enterotoxigenic Escherichia coli (ETEC) K88-induced diarrhea in piglets, but which bacteria play a key role and the mitigation mechanism have not been fully clarified. In this study, 36 male mice were randomly divided into six groups (CON, K88, BK [...] Read more.

Microecological preparation could relieve Enterotoxigenic Escherichia coli (ETEC) K88-induced diarrhea in piglets, but which bacteria play a key role and the mitigation mechanism have not been fully clarified. In this study, 36 male mice were randomly divided into six groups (CON, K88, BK (Bifidobacterium longum + K88), LK (Lactobacillus plantarum + K88), PK (Pediococcus acidilactici + K88), and MK (mixed strains + K88)) to explore the prevention mechanisms. Three probiotic strains and their mixtures (TPSM) significantly relieved the weight loss and restored the ratio of villus height to crypt depth in the jejunum. Except for Bifidobacterium longum, other strains significantly decreased interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in mice serum. The TPSM treatment significantly downregulated the mRNA expression of the inflammatory cytokines and the Toll-like receptor and downstream gene (TLR4, MyD88, NF-κB) in jejunum induced by ETEC. Furthermore, the TPSM could restore dysbiosis of the intestinal microbiota caused by ETEC. The intestinal microbiota analysis demonstrated that Bifidobacterium longum enriched the Bifidobacterium genus (p < 0.05), Lactobacillus plantarum enriched the Lactobacillus genus (p < 0.05), Pediococcus acidilactici enriched the Coriobacteriaceae_UCG-002 and Christensenellaceae_R-7_group genus (p < 0.05), mixed bacteria enriched the Akkermansia genus (p < 0.05), but ETEC enriched the Desulfovibrio genus (p < 0.05). Meanwhile, the starch and sucrose metabolism, galactose and fructose metabolism, mannose metabolism and ABC transporters were increased with probiotics pre-treatment (p < 0.05). To sum up, the microecological preparation alleviated ETEC-induced diarrhea by regulating the immune response, rebalancing intestinal microbiota and improving carbohydrate metabolism. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Animal design. Full article ">Figure 2
Effect of probiotics on the symptoms of ETEC K88-induced mice. (A) Before K88 inoculation (day 14), (B) after K88 inoculation (day 16), (C) spleen index, (D) colon length, (E) jejunal morphology (scale bars: 100 μm), (F) villus height/crypt depth. * represents significant differences vs. K88 group (*: p < 0.05, **: p < 0.01, ***: p < 0.001) # represents significant differences vs. CON group (#: p < 0.05,). All data are presented as the mean ± SD. Full article ">Figure 3
Effects of probiotics on inflammatory cytokines and immunoglobulin in serum of mice. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) IgA, (E) IgG, and (F) IgM. * represents significant differences vs. K88 group (*: p < 0.05, **: p < 0.01, ***: p < 0.001) # represents significant differences vs. CON group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001). All data are presented as the mean ± SD. Full article ">Figure 4
Effects of probiotics on inflammatory cytokines and Toll-like receptor mRNA expression in jejunum of mice. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) TLR4, (E) MyD88, (F) NF-κB. * represents significant differences vs. K88 group (***: p < 0.001) # represents significant differences vs. CON group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001). All data are presented as the mean ± SD. Full article ">Figure 5
Effect of probiotics on the diversity of intestinal microbiota. (A) Rarefaction curves, (B) Chao index, (C) Shannon index, (D) Simpson index, (E) PCoA. * represents significant differences vs. K88 group (*: p < 0.05, **: p < 0.01, ***: p < 0.001). Full article ">Figure 6
Effect of probiotics on intestinal microbiota composition. (A) Microbial distribution at the phylum level, (B) species difference analysis at genus level, (C) distribution histogram based on LDA, with a log LDA score above 3.0. Significant taxa are labeled and annotated with tags in the right panel. (D) Relative abundance of Lactobacillus, Desulfovibrio, Lachnospiraceae_NK4A136_group, Bifidobacterium, Enterorhabdus and Clostridium_sensu_stricto_1. * represents significant differences between the groups (*: p < 0.05, **: p < 0.01). Full article ">Figure 7
Dynamic bacterial functional profiles analyzed by PICRUSt (n = 6). (A) COG function classification, (B) metabolic pathways in level 1, (C) KEGG ortholog functional predictions (Level 2), (D) KEGG ortholog functional predictions of the relative abundances of top 25 metabolic functions (Level 3). * Means p < 0.05 for CON, BK, LK, PK, MK vs. K88. a, b Means with a row with different superscripts significantly differ (p < 0.05). Full article ">

16 pages, 3233 KiB

Open AccessArticle

The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites

by Kae Tomotsune, Fernanda Raya Tonetti, Hiroya Mizuno, Mariano Elean, Kohtaro Fukuyama, Binghui Zhou, Wakako Ikeda-Ohtsubo, Keita Nishiyama, Akihiro Yamamura, Hideaki Karasawa, Shinobu Ohnuma, Akira Horii, Tadao Saito, Haruki Kitazawa and Julio Villena

Int. J. Mol. Sci. 2022, 23(22), 14357; https://doi.org/10.3390/ijms232214357 - 18 Nov 2022

Cited by 4 | Viewed by 2199

Abstract

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout [...] Read more.

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3⁺NK1.1⁺CD8αα⁺) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-β and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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14 pages, 3090 KiB

Open AccessArticle

Immunostimulatory Activity of Lactic Acid Bacteria Cell-Free Supernatants through the Activation of NF-κB and MAPK Signaling Pathways in RAW 264.7 Cells

by Jaekoo Lee, Seonyoung Kim and Chang-Ho Kang

Microorganisms 2022, 10(11), 2247; https://doi.org/10.3390/microorganisms10112247 - 13 Nov 2022

Cited by 24 | Viewed by 3516

Abstract

Lactic acid bacteria (LAB) can improve host health and has strong potential for use as a health functional food. Specific strains of LAB have been reported to exert immunostimulatory effects. The primary goal of this study was to evaluate the immunostimulatory activities of [...] Read more.

Lactic acid bacteria (LAB) can improve host health and has strong potential for use as a health functional food. Specific strains of LAB have been reported to exert immunostimulatory effects. The primary goal of this study was to evaluate the immunostimulatory activities of novel LAB strains isolated from humans and foods and to investigate the probiotic properties of these strains. Cell-free supernatants (CFS) obtained from selected LAB strains significantly increased phagocytosis and level of nitric oxide (NO) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW264.7 macrophage cells. The protein expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, which are immunomodulators, was also upregulated by CFS treatment. CFS markedly induced the phosphorylation of nuclear factor-κB (NF-κB) and MAPKs (ERK, JNK, and p38). In addition, the safety of the LAB strains used in this study was demonstrated by hemolysis and antibiotic resistance tests. Their stability was confirmed under simulated gastrointestinal conditions. Taken together, these results indicate that the LAB strains selected in this study could be useful as probiotic candidates with immune-stimulating activity. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Effect of CFS from LAB strains on the phagocytosis of RAW264.7 macrophage cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h, and phagocytosis was evaluated by adding neutral red dye for 2 h. (a) The morphology of the untreated (control) or treated cells was visualized by phase-contrast light microscopy at a 20× magnification. (b) Phagocytic activity was quantified by measuring the logarithmic intensity using a microplate reader. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test. Full article ">Figure 2
Effect of CFS from LAB strains on the protein expression levels of iNOS and COX-2 in RAW264.7 cells. Representative Western blot and expression of iNOS (a,c) and COX-2 (b,d). Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The protein expression levels were quantified by densitometry, and the data were normalized relative to the intensity of β-actin. Untreated cells (control) were treated with DMEM only. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test. Full article ">Figure 3
Effect of CFS from LAB strains on cytokine production in RAW264.7 cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The amounts of TNF-α (a) and IL-6 (b) secreted by cells were measured using an ELISA kit. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test. Full article ">Figure 4
Effect of CFS from LAB strains on NF-κB activation in RAW264.7 cells. Representative Western blot (a) and activation levels of NF-κB (b). Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The NF-κB activation levels were quantified using densitometry. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test. Full article ">Figure 5
Effect of CFS from LAB strains on MAPK activation in RAW264.7 cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. Representative Western blot (a) and the activation levels of MAPK; ERK (b), JNK (c), and p38 (d) were quantified by densitometry. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test. Full article ">Figure 6
Hemolysis of LAB strains. (a) L. reuteri MG5462, (b) Lc. lactis MG4668, (c) Lc. lactis MG5278, (d) Lc. lactis MG5474, (e) L. fermentum MG4263, (f) L. fermentum MG4268, and (g) L. fermentum MG4282. Full article ">

16 pages, 2782 KiB

Open AccessArticle

Efficacy of Single and Multi-Strain Probiotics on In Vitro Strain Compatibility, Pathogen Inhibition, Biofilm Formation Capability, and Stress Tolerance

by Puvaneswari Puvanasundram, Chou Min Chong, Suriana Sabri, Md Sabri Mohd Yusoff, Keng Chin Lim and Murni Karim

Biology 2022, 11(11), 1644; https://doi.org/10.3390/biology11111644 - 10 Nov 2022

Cited by 8 | Viewed by 3975

Abstract

Compatibility of each strain in a multi-strain probiotic (MSP), along with its properties, becomes a strong base for its formulation. In this study, single-strain probiotics (SSPs) and multi-strain probiotics (MSPs) were evaluated in vitro for strain compatibility, microbial antagonism, biofilm formation capacity, and [...] Read more.

Compatibility of each strain in a multi-strain probiotic (MSP), along with its properties, becomes a strong base for its formulation. In this study, single-strain probiotics (SSPs) and multi-strain probiotics (MSPs) were evaluated in vitro for strain compatibility, microbial antagonism, biofilm formation capacity, and stress tolerance. Bacillus amyloliquefaciens L11, Enterococcus hirae LAB3, and Lysinibacillus fusiformis SPS11 were chosen as MSP1 candidates because they showed much stronger antagonism to Aeromonas hydrophila and Streptococcus agalactiae than a single probiotic. MSP 2 candidates were Lysinibacillus fusiformis strains SPS11, A1, and Lysinibacillus sphaericus strain NAS32 because the inhibition zone produced by MSP 2 against Vibrio harveyi and Vibrio parahaemolyticus was much higher than that produced by its constituent SSPs. MSP1 in the co-culture assay reduced (p < 0.05) A. hydrophila count from 9.89 ± 0.1 CFU mL⁻¹ to 2.14 ± 0.2 CFU mL⁻¹. The biofilm formation of both MSPs were significantly higher (p < 0.05) than its constituent SSPs and the pathogens. The SSPs in both MSPs generally showed resistance to high temperatures (80, 90, and 100 °C) and a wide range of pH (2 to 9). This in vitro assessment study demonstrates that MSP1 and 2 have the potential to be further explored as multi-strain probiotics on selected aquatic species. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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15 pages, 2070 KiB

Open AccessArticle

Oral Administration of Recombinant Lactoferrin-Expressing Probiotics Ameliorates Diet-Induced Lipid Accumulation and Inflammation in Non-Alcoholic Fatty Liver Disease in Mice

by Zhen-Shu Liu, Pei-Lin Li, Yu-We Ku and Po-Wen Chen

Microorganisms 2022, 10(11), 2215; https://doi.org/10.3390/microorganisms10112215 - 9 Nov 2022

Cited by 10 | Viewed by 2354

Abstract

We have recently developed probiotics that can express bovine, human, or porcine lactoferrin (LF), and the present study evaluated the effect of these probiotics in improving non-alcoholic fatty liver disease (NAFLD). Three kinds of probiotic supplements, including lactic acid bacteria (LAB), LAB/LF, and [...] Read more.

We have recently developed probiotics that can express bovine, human, or porcine lactoferrin (LF), and the present study evaluated the effect of these probiotics in improving non-alcoholic fatty liver disease (NAFLD). Three kinds of probiotic supplements, including lactic acid bacteria (LAB), LAB/LF, and inactivated LAB/LF, were prepared. The LAB supplement was prepared from 10 viable LAB without recombinant LF-expression, the LAB/LF supplement was prepared from 10 viable probiotics expressing LF, and the inactivated LAB/LF supplement was prepared from 10 inactivated probiotics expressing LF. A model of obese/NAFLD mice induced by a high-fat diet was established, and the mice were randomly divided into four groups and fed with a placebo, LAB, LAB/LF, or inactivated LAB daily for four weeks via oral gavage. The body weight, food intake, organ weight, biochemistry, and hepatic histopathological alterations and severity scoring were measured. The results revealed that the obese mice fed with any one of the three probiotic mixtures prepared from recombinant probiotics for four weeks exhibited considerably improved hepatic steatosis. These findings confirmed the assumption that specific probiotic strains or LF supplements could help to control NAFLD, as suggested in previous reports. Our data also suggest that the probiotics and LFs in probiotic mixtures contribute differently to improving the efficacy against NAFLD, and the expressed LF content in probiotics may help to boost their efficacy in comparison with the original probiotic mixtures. Moreover, when these LF-expressing probiotics were further inactivated by sonication, they displayed better efficacies than the viable probiotics against NAFLD. This study has provided intriguing data supporting the potential of recombinant probiotics in improving hepatic steatosis. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Figure 1
Growth curve (weight gain) in diet-induced obese (DIO) mice treated with three types of probiotic mixture. The treatment groups are detailed in the legend of <a href="#microorganisms-10-02215-t001" class="html-table">Table 1</a>. Briefly, DIO mice were fed with a high-fat diet and further supplemented with one of three probiotic mixtures by oral gavage daily for about four weeks. The mice were weighed twice a week during the study. The data represent group means. * The weight gains of LAB/LF mice were significantly higher than that of the placebo group. a The weight gains of LAB/LF (inactivated) mice were significantly lower than that of the placebo group. b The weight gains of LAB/LF (inactivated) were significantly lower than that of the LAB/LF group. Full article ">Figure 2
Tissue weights of liver (A) and epididymal fat (B) in DIO mice treated with three types of probiotic mixture for four weeks. The treatments are described in the legend of <a href="#microorganisms-10-02215-t001" class="html-table">Table 1</a>. Results are presented as means ± SDs. a,b Means with different letters are significantly different (p < 0.05) according to Student’s t-test. Full article ">Figure 3
Representative images of histopathological examinations of the liver in DIO mice treated with a placebo or one of three probiotic mixtures. The groups are defined and explained in the legend of <a href="#microorganisms-10-02215-t001" class="html-table">Table 1</a>. Livers showed multifocal and severe fatty changes in the microvesicles and macrovesicles (arrow), which were graded severe/high in the placebo ((A–C), Mouse A2), and moderate in the LAB ((D–F), Mouse B5), LAB-LF ((G–I), Mouse C6), and inactivated LAB/LF ((J–L), Mouse D3) groups. Liver sections were stained with H & E; images at 40×, 100×, and 400× magnification are shown. Grade 4 fatty infiltration was observed in most mice in the placebo control group, while Grade 2 fatty infiltration was often observed in the mice fed with the LAB, LAB/LF, or inactivated LAB/LF supplements. Full article ">Figure 4
Histopathological evaluation of fatty livers in individual obese mice supplemented with a placebo or one of three probiotic mixtures. The groups are defined in the legend of <a href="#microorganisms-10-02215-t001" class="html-table">Table 1</a>. Placebo group (A1 to A8, n = 8); LAB group (B1 to B8, n = 8); LAB/LF group (C1 to C8, n = 8); LAB/LF (inactivated) group (D1 to D8, n = 8). The amount of fat in the liver was graded from 0 to 4 as explained in <a href="#microorganisms-10-02215-f001" class="html-fig">Figure 1</a>. Full article ">Figure 5
Lipid profile of total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) in DIO mice. The DIO mice were administered a placebo, LAB, LAB/LF or the inactivated LAB/LF probiotic supplements for four weeks daily by oral gavage. Values indicate as the mean ± SD. * p < 0.05, ** p < 0.01 compared among the groups. Full article ">

21 pages, 1667 KiB

Open AccessArticle

Lacticaseibacillus casei Strain Shirota Modulates Macrophage-Intestinal Epithelial Cell Co-Culture Barrier Integrity, Bacterial Sensing and Inflammatory Cytokines

by Andrew Foey, Neama Habil, Alex Strachan and Jane Beal

Microorganisms 2022, 10(10), 2087; https://doi.org/10.3390/microorganisms10102087 - 21 Oct 2022

Cited by 10 | Viewed by 3508

Abstract

Probiotic bacteria modulate macrophage immune inflammatory responses, with functional cytokine responses determined by macrophage subset polarisation, stimulation and probiotic strain. Mucosal macrophages exhibit subset functional heterogeneity but are organised in a 3-dimensional tissue, over-laid by barrier epithelial cells. This study aimed to investigate [...] Read more.

Probiotic bacteria modulate macrophage immune inflammatory responses, with functional cytokine responses determined by macrophage subset polarisation, stimulation and probiotic strain. Mucosal macrophages exhibit subset functional heterogeneity but are organised in a 3-dimensional tissue, over-laid by barrier epithelial cells. This study aimed to investigate the effects of the probiotic Lacticaseibacillus casei strain Shirota (LcS) on macrophage-epithelial cell cytokine responses, pattern recognition receptor (PRR) expression and LPS responses and the impacts on barrier integrity. THP-1-derived M1 and M2 subset macrophages were co-cultured in a transwell system with differentiated Caco-2 epithelial cells in the presence or absence of enteropathogenic LPS. Both Caco-2 cells in monoculture and macrophage co-culture were assayed for cytokines, PRR expression and barrier integrity (TEER and ZO-1) by RT-PCR, ELISA, IHC and electrical resistance. Caco-2 monocultures expressed distinct cytokine profiles (IL-6, IL-8, TNFα, endogenous IL-10), PRRs and barrier integrity, determined by inflammatory context (TNFα or IL-1β). In co-culture, LcS rescued ZO-1 and TEER in M2/Caco-2, but not M1/Caco-2. LcS suppressed TLR2, TLR4, MD2 expression in both co-cultures and differentially regulated NOD2, TLR9, Tollip and cytokine secretion. In conclusion, LcS selectively modulates epithelial barrier integrity, pathogen sensing and inflammatory cytokine profile; determined by macrophage subset and activation status. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Macrophage subset-epithelial cell co-culture and cytokine production determine barrier integrity. Caco-2 epithelial cells were cultured to confluence (intact barrier) in transwell inserts and incubated in co-culture with M1 and M2 Mφ subsets (a,b) or Mφ-derived cytokines in the absence of Mφs (c,d). Co-cultures received LPS in the apical compartment and cytokine production was analysed in the basolateral compartment (a) and the corresponding TEER as a measure of barrier integrity (b). Effect of Mφ-derived TNFα, IL-1β, IL-8 and IL-10 on barrier integrity is presented as TEER in Ω cm2 (c) and expression of the tight junction molecule, ZO-1 mRNAexpression (d) is presented as relative expression (RQ, Arbitary Units) compared to GAPDH housekeeping gene expression. Mφ co-culture and effect of inflammatory stimuli is further represented by immunohistochemical characterisation of ZO-1 protein staining in the epithelial barriers incubated in co-cultures (e–k), where (e) Caco-2 epithelial barrier control, (f) Caco-2 + M1 Mφs, (g) Caco-2 + M1 + LPS, (h) Caco-2 + M1 + IL-1β, (i) Caco-2 + M2 Mφs, (j) Caco-2 + M2 + LPS and (k) Caco-2 + M2 + IL-1β. Data displayed is a representative experiment with triplicate samples for n = 4 replicate experiments (a–d) and n = 3 experiments (e–k). Significant effects of Caco-2/Mφ co-culture are compared to Caco-2 control (b) and cytokine effects compared to unstimulated Caco-2 control (c,d) and significance indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 2
LcS fails to rescue barrier integrity in M1 Macrophage subset-epithelial cell co-culture. Caco-2 epithelial cells were cultured to confluence (intact barrier) in transwell inserts and incubated in co-culture with M1 (bold shading) and M2 (hatched shading) Mφ subsets in the presence (b,d) or absence (a,c) of apically applied LPS stimulation (100 ng/mL). Barrier integrity of co-cultures was analysed by measuring TEER (a,b) (with un-stimulated Caco-2 control barrier integrity TEER being indicated by dashed line) and presented in Ω cm2, and mRNA expression of the tight junction molecule, ZO-1 (c,d) is presented as relative expression (RQ, Arbitary Units) compared to GAPDH housekeeping gene expression. Data displayed is a representative experiment with triplicate samples for n = 3 replicate experiments. Significant effects of LcS treatment on Caco-2/Mφ co-cultures in the presence or absence of LPS stimulation are compared to non-LcS treated co-culture controls and significance indicated as * p < 0.05, *** p < 0.001 and ns, not significant and small-scale changes (d) are indicated by an arrow indicating augmentation/rescue and accompanied by the stated RQ relative expression values. Full article ">Figure 3
LcS differentially modulates bacterial sensing molecules in M1 & M2 Macrophage subset-epithelial cell co-culture. Caco-2 epithelial cells were cultured to confluence (intact barrier) in transwell inserts and incubated in co-culture with M1 (bold) and M2 (hatched) Mφ subsets in the presence of apically applied LPS stimulation (100 ng/mL) in the presence or absence of LcS. The LPS-receptor, TLR4, was measured as mRNA expression (a) and surface protein by flow cytometry (b). The mRNA expression of co-receptor molecules CD14 (c) and MD-2 (d) were also analysed, as was TLR2 mRNA (e), TLR2 surface protein (f), NOD2 mRNA (g), TLR9 mRNA (h) and the negative regulator of TLR signalling, Tollip mRNA (i). Gene expression is presented as relative expression (RQ, Arbitary Units) compared to GAPDH housekeeping gene expression and surface protein by flow cytometry as net MFI (Arbitary Units). Data displayed is a representative experiment with triplicate samples for n = 3 replicate experiments. Significant effects of LcS treatment on Caco-2/Mφ co-cultures in the presence of LPS stimulation are compared to non-LcS treated co-culture controls and significance indicated as * p < 0.05, *** p < 0.001 and ns, not significant and small-scale changes (i) are indicated by the stated RQ relative expression values. Full article ">Figure 4
LcS differentially modulates M1 & M2 co-culture cytokines: dependency on LPS stimulation. Caco-2 epithelial cells were cultured to confluence (intact barrier) in transwell inserts and incubated in co-culture with M1 (bold) and M2 (hatched) Mφ subsets in the presence (b,d,f,h) or absence (a,c,e,g) of apically applied LPS stimulation (100 ng/mL) in the presence or absence of apically applied LcS. The pro-inflammatory cytokines, TNFα (a,b), IL-6 (c,d), IL-8 (e,f) and anti-inflammatory cytokine, IL-10 (g,h) were measured by sandwich ELISA and presented as protein production of the same secreted cytokines as the mean ± SD in pg/mL. Data displayed is a representative experiment with triplicate samples for n = 3 replicate experiments. Significant effects of LcS are compared to either the unstimulated control or LPS-stimulated control and are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001 and ns, not significant and small scale changes (a,e,f) are indicated with arrow bars indicating augmentation or suppression and accompanied with an indication of cytokine concentration measured. Full article ">

19 pages, 4085 KiB

Open AccessArticle

Screening of Bacteria Inhibiting Clostridium perfringens and Assessment of Their Beneficial Effects In Vitro and In Vivo with Whole Genome Sequencing Analysis

by Zipeng Jiang, Weifa Su, Mingzhi Yang, Wentao Li, Tao Gong, Yu Zhang, Chaoyue Wen, Xinxia Wang, Yizhen Wang, Mingliang Jin and Zeqing Lu

Microorganisms 2022, 10(10), 2056; https://doi.org/10.3390/microorganisms10102056 - 18 Oct 2022

Cited by 7 | Viewed by 2949

Abstract

Various countries and organizations call for banning the use of antibiotic growth promoters (AGPs) as prophylaxis and for growth promotion in the livestock industry. Hence, seeking a substitute for antibiotics is strongly required by the livestock industry to maintain the productivity level and [...] Read more.

Various countries and organizations call for banning the use of antibiotic growth promoters (AGPs) as prophylaxis and for growth promotion in the livestock industry. Hence, seeking a substitute for antibiotics is strongly required by the livestock industry to maintain the productivity level and profits. Probiotics could represent one viable solution because of their beneficial effects on host health and maintaining the intestinal microbiota balance. In the present study, we aimed to isolate bacterial strains with probiotics properties from JinHua pig (a Chinese native pig breed) gastrointestinal tract that have antagonistic activity against to common disease-causing bacteria on farms. The four most potent strains were isolated (PP31, BA11, BA40, BV5) by the agar well diffusion method and further characterized by acid, bile salt, trypsin tolerance, whole genome sequencing (WGS), and suppressing Clostridium perfringens adhesion to IPEC-J2 cells. According to these results, BA40 had the highest number and variety of probiotic secondary metabolic secretion genes and capacity to exclude the attachment of Clostridium perfringens to IPEC-J2 cells as same as PB6. The animal experiment in vivo illustrated that BA40 and PB6 could reduce the phenomenon induced by Clostridium perfringens challenge of body weight loss, colon length decrease, pro-inflammatory cytokine increase, and Clostridium perfringens and Escherichia coli increase. The present study provides evidence that BA40 could represent a novel probiotic candidate as PB6, which exhibited some probiotic features and mitigated the burden of Clostridium perfringens associated gut disease. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Experimental design. Full article ">Figure 2
Agar well diffusion assay illustrating the growth inhibition of pathogenic bacteria by cell-free supernatants extracted from the isolated strains. (A) Escherichia coli plate. (B) Salmonella enterica plate. (C) Staphylococcus aureus plate. (D) Clostridium perfringens plate. (E) Inhibition zone diameter of Escherichia coli (F) Inhibition zone diameter of Salmonella enterica (G) Inhibition zone diameter of Staphylococcus aureus (H) Inhibition zone diameter of Clostridium perfringens. a, b, c Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions. Full article ">Figure 3
Probiotic properties of isolated strains. (A) The growth curves. (B) The ability of acid tolerance. (C) The ability of bile salt tolerance. (D) The ability of trypsin tolerance. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions. Full article ">Figure 4
The inhibitory results of isolate strains on the adhesion of Clostridium perfringens. a, b, c Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions. Full article ">Figure 5
Annotated chord diagrams based on database matching for classification. (A) Correspondence between annotated information on bacterial genomes and metabolic pathways obtained by KEGG database. (B) The COG database was compared to classify the predicted proteins into gene families and to give the corresponding functional annotation information for the family. Full article ">Figure 6
The protective effect of probiotic candidates against Clostridium perfringens infection in mice. (A) Bodyweight (BW). (B) At the end of experiment, mice weight. (C) The spleen index. (D) The liver index. (E) The colon images (F) The colon length. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained six repetitions. Partial data of this figure were published in previous study. Adapted with permission from ref. [<a href="#B29-microorganisms-10-02056" class="html-bibr">29</a>]. Copyright 2021 Jiang, Li, Su, Wen, Gong, Zhang, Wang, Jin and Lu. Full article ">Figure 7
The population of intestinal microbiota of mice. (A) Clostridium perfringens in the ileum. (B) Clostridium perfringens in the cecum. (C) Escherichia coli in the ileum. (D) Escherichia coli in the cecum. (E) Lactobacillus in the ileum. (F) Lactobacillus in the cecum. Results are presented as mean ± SD (The data were presented as log10 gene copies/g of intestinal digesta). a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). Full article ">Figure 8
The effect of isolated probiotics treatment on inflammatory cytokines, immunoglobulin, DAO and DLA in mice. (A) IL-1β concentrations. (B) IL-6 concentrations. (C) TNF-α concentrations. (D) IgA concentrations. (E) IgG concentrations. (F) sIgA concentrations. (G) iNOS concentrations. (H) NO concentrations. (I) DAO concentrations. (J) DLA concentrations. Results are presented as mean ± SD. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained six repetitions. Full article ">

10 pages, 1476 KiB

Open AccessArticle

A Heat-Killed Probiotic Mixture Regulates Immune T Cells Balance and IgE Production in House Dust Mite Extraction-Induced Atopic Dermatitis Mice

by Hsin-Yu Chen, Yung-Tsung Chen, Kuan-Yi Li, Hsiao-Wen Huang, Yu-Chun Lin and Ming-Ju Chen

Microorganisms 2022, 10(10), 1881; https://doi.org/10.3390/microorganisms10101881 - 21 Sep 2022

Cited by 2 | Viewed by 3312

Abstract

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease accompanied with severe itching and skin lesions. Current studies have demonstrated that probiotics can exert an immunomodulatory effect, improve epithelial barrier function, and normalize the composition of gut microbiota. Thus, the aim [...] Read more.

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease accompanied with severe itching and skin lesions. Current studies have demonstrated that probiotics can exert an immunomodulatory effect, improve epithelial barrier function, and normalize the composition of gut microbiota. Thus, the aim of this study was to investigate the effect of probiotics on the immune balance of AD in vivo. We first screened two lactic acid bacteria strains, which were Lactococcus lactis subsp. cremoris MP01 and Lactobacillus paracasei subsp. paracasei MP02, from 10 strains isolated from traditional fermented milk with inflammation regulating activities in vitro. In the house dust mite (HDM) extraction-induced AD mouse model, mice were assigned randomly to four groups: control group (PC), HDM-induced AD group (NC), HDM-induced AD mice with administration of a mixture of heat-killed MP01 and MP02 at a low concentration (LD), and high concentration (HD) groups. Compared with the NC group, the probiotic treatments could relieve the AD symptoms. Moreover, the LD group significantly decreased total and HDM-specific IgE concentration. These results indicated that a combination of heat-killed MP01 and MP02 strains modulated the proportion of IL4⁺CD4⁺ T cells and IFNγ⁺CD4⁺ T cells in the spleen of HDM extraction-induced AD mice. In conclusion, administration of the heat-killed MP01 and MP02 mixtures appeared to relieve the classic AD signs, decrease serum IgE concentration, and rebalance the population of Th1/Th2 cells in HDM extraction-induced AD mice. The immunomodulatory activities of a combination of heat-killed MP01 and MP02 provided a potential new therapeutic strategy against AD. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Effects of the different probiotics on the production of TNF-α (a) and IL10 (b) after 24 h co-culture with RAW 264.7. NC: negative control; PC: positive control (50 ng/mL LPS). Data were analyzed by ANOVA with Duncan’s multiple comparison tests. Means for each group without a common letter are significantly different (p < 0.05). Value represents means ± SD (n = 3). Full article ">Figure 2
A combination of heat-killed MP01 and MP02 strains alleviated skin lesions of HDM-extraction induced AD mice. (a) The body weight of mice after treatment for 46 days. Value represents means ± SD. (n = 7–10/group) (b) The skin lesion induced by the HDM extraction in NC, PC, LD, and HD groups. Aspects of dermatitis in AD mice were examined on day 45. (c) Histological section of skin of the HDM-induced AD in NC, PC, LD, and HD groups. The dorsal skin from mice was stained by hematoxylin and eosin stain, and the slides were examined under 400× magnification. Full article ">Figure 3
A combination of heat-killed MP01 and MP02 strains rebalanced the proportion of Th1 and Th2 cells in the spleen of HDM-extraction-induced AD mice. (a) The spleen weight of mice after treatment for 46 days. The population of (b) IFN-γ+CD4+ T cells, (c) IL4+CD4+ T cells, and (d) CD4+CD25+Foxp3+ T cells in the spleen of test mice. The columns and the error bars represent means ± SD (n = 7–10/group). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. Value represents means ± SD. Full article ">Figure 4
A combination of heat-killed MP01 and MP02 strains decreased the level of HDM-specific IgE (a,b) in HDM-extraction induced AD mice. (n = 7–10/group) Data were analyzed using an ANOVA with Dunnett’s multiple comparison tests. * p < 0.05, **** p < 0.0001. Value represents means ± SD. Full article ">

14 pages, 5939 KiB

Open AccessArticle

Anti-Tumor Effect of Heat-Killed Bifidobacterium bifidum on Human Gastric Cancer through Akt-p53-Dependent Mitochondrial Apoptosis in Xenograft Models

by SukJin Kim, Hwan Hee Lee, Wonhyeok Choi, Chang-Ho Kang, Gun-Hee Kim and Hyosun Cho

Int. J. Mol. Sci. 2022, 23(17), 9788; https://doi.org/10.3390/ijms23179788 - 29 Aug 2022

Cited by 17 | Viewed by 3595

Abstract

Paraprobiotics, inactivated microbial cells, regulate immune system and exhibit antioxidant and anti-inflammatory activities in patients with weakened immunity or the elderly. This study evaluated the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus on human gastric cancer MKN1 cells in vitro and in vivo [...] Read more.

Paraprobiotics, inactivated microbial cells, regulate immune system and exhibit antioxidant and anti-inflammatory activities in patients with weakened immunity or the elderly. This study evaluated the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus on human gastric cancer MKN1 cells in vitro and in vivo in xenograft animal models. First, cytotoxicity and apoptosis in MKN1 cells of 11 different heat-killed Bifidobacterium or Lactobacillus strains were examined using the MTT assay or flow cytometry, respectively. Then, BALB/c nude mice xenograft animal models were implanted with human gastric cancer MKN1 cells and orally administered a selected single or a mixture of heat-killed bacterial strains to investigate their inhibitory effect on tumor growth. In addition, the expression of p-Akt, p53, Bax, Bak, cleaved caspase-9, -3, and PARP in the tumor tissues was analyzed using Western blotting assay or immunohistochemistry staining. The results show that heat-killed B. bifidum MG731 (MG731), L. reuteri MG5346 (MG5346), and L. rhamnosus MG5200 (MG5200) induced relatively greater apoptosis than other strains in MKN1 cells. Oral administration of a single dose or a mixture of MG731, MG5346, or MG5200 significantly delayed tumor growth, and MG731 had the most effective anti-tumor effect in the xenograft model. Protein expression of p-Akt, p53, Bax, cleaved caspase-3 and -9, and PARP in tumors derived from the xenograft model correlated with the results of the immunohistochemistry staining. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Cytotoxicity of heat-killed Bifidobacterium and Lactobacillus in human gastric cancer MKN1 cells analyzed by MTT assay. (a) Cell morphology and (b) cell viability (%). Results are presented as mean ± SD in three independent experiments. * p < 0.05 compared with the control. Full article ">Figure 2
Apoptosis of heat-killed Bifidobacterium and Lactobacillus in human gastric cancer MKN1 cells analyzed by flow cytometry assay. (a) Representative Annexin V and PI staining for apoptosis and (b) total apoptosis (%) in MKN1 cells. Results are presented as mean ± SD in three independent experiments. * p < 0.05 compared with the control. Full article ">Figure 3
The tumor growth inhibitory effect of heat- killed Bifidobacterium and Lactobacillus in xenograft model implanted with human gastric cancer MKN1 cells. BALB/c nude mice were subcutaneously injected with 5 × 106 MKN1 cells/mouse into the dorsum next to right hind leg. The mice were orally administrated drinking water or a single or mixed strain of heat-killed bacterial strains every day for another 19 days. (a) Photographs represent tumor size of xenograft model mice. (b) Tumor growth curve. (c) Tumor weight (mg). (d) Body weight changes. Results are presented as the mean ± SD (n = 6). * p < 0.05 compared with the control group. Control, drinking water; MG731, B. bifidum MG731 (1 × 109 cells/mouse); MG5346, L. reuteri MG5346 (1 × 109 cells/mouse); MG4584, L. rhamnosus MG5200 (1 × 109 cells/mouse); 3Mix-1, 1:1:1 mixture of B. bifidum MG731 + L. reuteri MG5346 + L. rhamnosus MG5200 (1 × 109 cells/mouse); 3Mix-3, mixture of 3Mix-3, 1:1:1 mixture of B. bifidum MG731 + L. reuteri MG5346 + L. rhamnosus MG5200 (3 × 109 cells/mouse). Full article ">Figure 4
Expression of apoptosis-related proteins in tumor tissues from xenograft model analyzed by Western blotting. After 19 days of administration, proteins were extracted from the tumor tissues and subjected to Western blotting. (a) The representative Western blot images; Western blot quantitative data of (b) p-Akt/Akt, (c) p53/GAPDH, (d) Bax/GAPDH, and (e) Bak/GAPDH. Results are presented as the mean ± SD (n = 6). * p < 0.05 compared with the control group. Full article ">Figure 5
Expression of apoptosis-related proteins in tumor tissues from xenograft model analyzed by Western blotting assay. After 19 days of heat-killed single or mixed strain treatment, proteins were extracted from the tumor tissues and subjected to Western blotting. (a) The representative Western blot images; Western blot quantitative data of (b) cleaved caspase-9/caspase-9, (c) cleaved caspase-3/caspase-3, and (d) cleaved PARP/PARP. Results are presented as the mean ± SD (n = 6). * p < 0.05 compared with the control group. Full article ">Figure 6
Immunohistochemistry analysis of the tumor tissues. After 19 days of heat-killed single or mixed strain treatment, tumor tissues were harvested and stained by (a) p-Akt, (b) p53, (c) Bax, (d) cleaved caspase-9, (e) cleaved caspase-3, and (f) cleaved PARP. The scale bar represents 100 px. Brown dots are expression of p-Akt, p53, Bax, cleaved caspase-9 and -3, PARP. Black arrows indicate the brown dots of IHC (magnification 200× or 400× as indicated). Full article ">

13 pages, 2651 KiB

Open AccessArticle

Limosilactobacillus reuteri Attenuates Atopic Dermatitis via Changes in Gut Bacteria and Indole Derivatives from Tryptophan Metabolism

by Zhifeng Fang, Tong Pan, Hongchao Wang, Jinlin Zhu, Hao Zhang, Jianxin Zhao, Wei Chen and Wenwei Lu

Int. J. Mol. Sci. 2022, 23(14), 7735; https://doi.org/10.3390/ijms23147735 - 13 Jul 2022

Cited by 30 | Viewed by 3827

Abstract

Gut bacteria are closely associated with the development of atopic dermatitis (AD) due to their immunoregulatory function. Indole derivatives, produced by gut bacteria metabolizing tryptophan, are ligands to activate the aryl hydrocarbon receptor (AHR), which plays a critical role in attenuating AD symptoms. [...] Read more.

Gut bacteria are closely associated with the development of atopic dermatitis (AD) due to their immunoregulatory function. Indole derivatives, produced by gut bacteria metabolizing tryptophan, are ligands to activate the aryl hydrocarbon receptor (AHR), which plays a critical role in attenuating AD symptoms. Limosilactobacillus reuteri, a producer of indole derivatives, regulates mucosal immunity via activating the AHR signaling pathway. However, the effective substance and mechanism of L. reuteri in the amelioration of AD remain to be elucidated. In this research, we found that L. reuteri DYNDL22M62 significantly improved AD-like symptoms in mice by suppressing IgE levels and the expressions of thymic stromal lymphopoietin (TSLP), IL-4, and IL-5. L. reuteri DYNDL22M62 induced an increase in the production of indole lactic acid (ILA) and indole propionic acid (IPA) via targeted tryptophan metabolic analysis and the expression of AHR in mice. Furthermore, L. reuteri DYNDL22M62 increased the proportions of Romboutsia and Ruminococcaceae NK4A214 group, which were positively related to ILA, but decreased Dubosiella, which was negatively related to IPA. Collectively, L. reuteri DYNDL22M62 with the role of modulating gut bacteria and the production of indole derivatives may attenuate AD via activating AHR in mice. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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Figure 1
Effects of Limosilactobacillus reuteri on pathological symptoms of AD-like mice. (A) Experimental design. (B) Effects of L. reuteri strains on ear thickness of AD-like mice. (C) H&E staining of skin lesions, scale bar = 200 μm, original magnification = 400×. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. DNFB group. Full article ">Figure 2
L. reuteri strains regulated IgE levels and the expressions of Th2 cytokines in AD-like mice. (A) IgE levels. (B–D) Changes in TSLP, IL-4, and IL-5 levels. * p < 0.05, ** p < 0.01 vs. DNFB group, ns, no significance. TSLP, thymic stromal lymphopoietin. Full article ">Figure 3
L. reuteri strains affected indole derivatives levels and the expression of AHR. (A–E) Changes in ILA, IAld, IAA, IA, and IPA levels in fecal samples. (F) Fold-change of AHR expression. * p < 0.05, ** p < 0.01 vs DNFB group, ns, no significance. ILA, indolelactic acid, IAld, indole-3-aldehyde, IAA, indoleacetic acid, IA, indoleacrylic acid, IPA, indole propionic acid. Full article ">Figure 4
L. reuteri strains regulated gut microbiota in AD-like mice. (A) PCA plot between groups. (B) Gut microbial composition at the phylum level. (C) Effects of L. reuteri DYNDL22M62 on gut microbial composition. * p < 0.05, ** p < 0.01, *** p < 0.001 vs DNFB group. PCA, principal component analysis, PC1, principal component 1, PC2, principal component 2. Full article ">Figure 5
Differential gut bacteria and the correlation with indole derivatives. (A) Differential bacteria between groups using LEfSe analysis. (B) Comparison of differential bacteria. (C) The correlation between differential bacteria and indole derivatives (95% confidence interval, red cross, no significance). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DNFB group. LEfSe, linear discriminant analysis (LDA) effect size. Full article ">

12 pages, 684 KiB

Open AccessArticle

Machine Learning Algorithms Highlight tRNA Information Content and Chargaff’s Second Parity Rule Score as Important Features in Discriminating Probiotics from Non-Probiotics

by Carlo M. Bergamini, Nicoletta Bianchi, Valerio Giaccone, Paolo Catellani, Leonardo Alberghini, Alessandra Stella, Stefano Biffani, Sachithra Kalhari Yaddehige, Tania Bobbo and Cristian Taccioli

Biology 2022, 11(7), 1024; https://doi.org/10.3390/biology11071024 - 7 Jul 2022

Cited by 5 | Viewed by 2727

Abstract

Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four [...] Read more.

Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four Machine Learning algorithms were employed to accurately identify probiotic bacteria based on their DNA characteristics. Although the prediction accuracies of all algorithms were excellent, the Neural Network returned the highest scores in all the evaluation metrics, managing to discriminate probiotics from non-probiotics with an accuracy greater than 90%. Interestingly, our analysis also highlighted the information content of the tRNA sequences as the most important feature in distinguishing the two groups of organisms probably because tRNAs have regulatory functions and might have allowed probiotics to evolve faster in the human gut environment. Through the methodology presented here, it was also possible to identify seven promising new probiotics that have a higher information content in their tRNA sequences compared to non-probiotics. In conclusion, we prove for the first time that Machine Learning methods can discriminate human probiotic from non-probiotic organisms underlining information within tRNA sequences as the most important genomic feature in distinguishing them. Full article

(This article belongs to the Topic Application of Probiotics and Their Potential Health Benefits)

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