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Microencapsulation of probiotic bacteria Lactobacillus plantarum 15HN using alginate-psyllium-fenugreek polymeric blends

Haghshenas B, Abdullah N, Nami Y, Radiah D, Rosli R, Yari Khosroushahi A

J Appl Microbiol, 2015 Apr;118(4):1048-57.
PMID: 25619628 DOI: 10.1111/jam.12762

Investigation on the use of herbal-based biopolymers for probiotic-Lactobacillus plantarum 15HN-encapsulation is presented. The objectives are to enhance its oral delivery, colonic release and survival rate of these probiotic cultures in gastrointestinal environment.
A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells

Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Yari Khosroushahi A

J Appl Microbiol, 2014 Aug;117(2):498-508.
PMID: 24775273 DOI: 10.1111/jam.12531

This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract.
Fulltext Effect of psyllium and gum Arabic biopolymers on the survival rate and storage stability in yogurt of Enterococcus duransIW3 encapsulated in alginate

Nami Y, Haghshenas B, Yari Khosroushahi A

Food Sci Nutr, 2017 05;5(3):554-563.
PMID: 28572941 DOI: 10.1002/fsn3.430

Different herbal biopolymers were used to encapsulate Enterococcus durans IW3 to enhance its storage stability in yogurt and subsequently its endurance in gastrointestinal condition. Nine formulations of encapsulation were performed using alginate (ALG), ALG-psyllium (PSY), and ALG-gum Arabic (GA) blends. The encapsulation efficiency of all formulations, tolerance of encapsulated E. durans IW3 against low pH/high bile salt concentration, storage lifetime, and release profile of cells in natural condition of yogurt were evaluated. Result revealed 98.6% encapsulation efficiency and 76% survival rate for all formulation compared with the unencapsulated formulation cells (43%). The ALG-PSY and ALG-GA formulations have slightly higher survival rates at low pH and bile salt condition (i.e., 76-93% and 81-95%, respectively) compared with the ALG formulation. All encapsulated E. durans IW3 was released from the prepared beads of ALG after 90 min, whereas both probiotics encapsulated in ALG-GA and ALG-PSY were released after 60 min. Enterococcus durans IW3 was successfully encapsulated in ALG, ALG-GA, and ALG-PSY beads prepared by extrusion method. ALG-GA and ALG-PSY beads are suitable delivery carriers for the oral administration of bioactive compounds like probiotics. The GA and PSY gels exhibited better potential for encapsulation of probiotic bacteria cells because of the amendment of ALG difficulties and utilization of therapeutic and prebiotic potentials of these herbal biopolymers.
Herbal hydrogel-based encapsulated Enterococcus faecium ABRIINW.N7 improves the resistance of red hybrid tilapia against Streptococcus iniae

Kahieshesfandiari M, Nami Y, Lornezhad G, Kiani A, Javanmard A, Jaymand M, et al.

J Appl Microbiol, 2021 Nov;131(5):2516-2527.
PMID: 33817937 DOI: 10.1111/jam.15098

AIMS: The streptococcal disease has been associated with serious mortality and significant global economic loss in the tilapia farming industry. The overall goal of this work was to test herbal hydrogels based on encapsulated Enterococcus faecium ABRIINW.N7 for potential probiotic anti-microbial activity against Streptococcus iniae in red hybrid tilapia.
METHODS AND RESULTS: Abnormal behaviour, clinical signs, postinjection survival and histopathology (kidney, liver, eye and brain) were measured. Cumulative mortality of CON+ , free cells, ALG and treatments (F1-F7) was 30, 24, 22, 19, 17, 17, 16, 14, 14 and 12 out of 30 fish and the survival rates for E. faecium ABRIINW.N7 microencapsulated in an alginate-BS blend with 0·5, 1, 1·5, 2, 2·5 and 3% fenugreek were 43, 43, 47, 53, 53 and 60%, respectively. After the incorporation of fenugreek with the alginate-BS blend, there was an 8-21% increase in probiotic cell viability. Furthermore, the survival rate for the alginate-BS blend with 2·5 and 3% fenugreek (F6 and F7) was significantly (P ≤ 0·05) higher than other blends. The highest encapsulation efficiency, viability in gastrointestinal conditions and during storage time and excellent antipathogenicity against S. iniae were observed in alginate-BS +3% fenugreek formulation (F7).
CONCLUSIONS: It is recommended that probiotic strains like E. faecium ABRIINW.N7 in combination with local herbal gums, such as BS and fenugreek plus alginate, can be used as a suitable scaffold and an ideal matrix for the encapsulation of probiotics.
SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes models connecting process parameters, matrix structure and functionality.
Fulltext The Prophylactic Effect of Probiotic Enterococcus lactis IW5 against Different Human Cancer Cells

Nami Y, Haghshenas B, Haghshenas M, Abdullah N, Yari Khosroushahi A

Front Microbiol, 2015;6:1317.
PMID: 26635778 DOI: 10.3389/fmicb.2015.01317

Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, MCF-7, AGS, HT-29, and Caco-2. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications.
Fulltext Antimicrobial activity and the presence of virulence factors and bacteriocin structural genes in Enterococcus faecium CM33 isolated from ewe colostrum

Nami Y, Haghshenas B, Haghshenas M, Yari Khosroushahi A

Front Microbiol, 2015;6:782.
PMID: 26284059 DOI: 10.3389/fmicb.2015.00782

Screening of lactic acid bacteria (LAB) isolated from ewe colostrum led to the identification and isolation of Enterococcus faecium CM33 with interesting features like high survival rates under acidic or bile salts condition, high tolerance for the simulated gastrointestinal condition, and high adhesive potential to Caco-2 cells. According the inhibition of pathogen adhesion test results, this strain can reduce more than 50% adhesion capacity of Escherichia coli, Shigella flexneri, Klebsiella pneumoniae, Listeria monocytogenes, and Staphylococcus aureus to Caco-2 cells. Based on the antibiotic sensitivity test findings, E. faecium CM33 was susceptible to gentamycin, vancomycin, erythromycin, ampicillin, penicillin, tetracycline, and rifampicin, but resistant to chloramphenicol, clindamycin, and kanamycin. Upon assessment of the virulence determinants for E. faecium CM33, this strain was negative for all tested virulence genes. Furthermore, the genome of this strain was evaluated for the incidence of the known enterocin genes by specific PCR amplification and discovered the genes encoding enterocins A, 31, X, and Q. Based on this study findings, the strain E. faecium CM33 can be considered as a valuable nutraceutical and can be introduced as a new potential probiotic.
Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines

Haghshenas B, Abdullah N, Nami Y, Radiah D, Rosli R, Khosroushahi AY

Anaerobe, 2014 Dec;30:51-9.
PMID: 25168457 DOI: 10.1016/j.anaerobe.2014.08.009

Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 μg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics.
Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells

Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Khosroushahi AY

Anaerobe, 2014 Aug;28:29-36.
PMID: 24818631 DOI: 10.1016/j.anaerobe.2014.04.012

Lactobacillus acidophilus is categorized as a probiotic strain because of its beneficial effects in human health and prevention of disease transmission. This study is aimed to characterize the probiotic potential of L. acidophilus 36YL originally isolated from the vagina of healthy and fertile Iranian women. The L. acidophilus 36YL strain was identified using 16S rDNA gene sequencing and characterized by biochemical methodologies, such as antibiotics susceptibility, antimicrobial activity, and acid and bile resistance. The bioactivity of the secretion of this strain on four human cancer cell lines (AGS, HeLa, MCF-7, and HT-29) and one normal cell line (HUVEC) was evaluated by cytotoxicity assay and apoptosis analysis. This newly isolated strain was found to exhibit notable probiotic properties, such as admirable antibiotic susceptibility, good antimicrobial activity, and favorable resistance to acid and bile salt. The results of bioactivity assessment demonstrated acceptable anticancer effects on the four tested cancer cell lines and negligible side effects on the assayed normal cell line. Our findings revealed that the anticancer effect of L. acidophilus 36YL strain secretions depends on the induction of apoptosis in cancer cells. L. acidophilus 36YL strain is considered as a nutraceutical alternative or a topical medication with a potential therapeutic index because of the absence of cytotoxicity to normal cells, but effective toxicity to cancer cell lines.
Fulltext Bioactivity characterization of Lactobacillus strains isolated from dairy products

Haghshenas B, Nami Y, Haghshenas M, Abdullah N, Rosli R, Radiah D, et al.

Microbiologyopen, 2015 Oct;4(5):803-13.
PMID: 26219634 DOI: 10.1002/mbo3.280

This study aimed to find candidate strains of Lactobacillus isolated from sheep dairy products (yogurt and ewe colostrum) with probiotic and anticancer activity. A total of 100 samples were randomly collected from yogurt and colostrum and 125 lactic acid bacteria were isolated. Of these, 17 Lactobacillus strains belonging to five species (L. delbrueckii, L. plantarum, L. rhamnosus, L. paracasei, and L. casei) were identified. L. plantarum 17C and 13C, which isolated from colostrums, demonstrated remarkable results such as resistant to low pH and high concentrations of bile salts, susceptible to some antibiotics and good antimicrobial activity that candidate them as potential probiotics. Seven strains (1C, 5C, 12C, 13C, 17C, 7M, and 40M), the most resistant to simulated digestion, were further investigated to evaluate their capability to adhere to human intestinal Caco-2 cells. L. plantarum 17C was the most adherent strain. The bioactivity assessment of L. plantarum 17C showed anticancer effects via the induction of apoptosis on HT-29 human cancer cells and negligible side effects on one human epithelial normal cell line (FHs 74). The metabolites produced by this strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells.
Probiotic assessment of Enterococcus durans 6HL and Lactococcus lactis 2HL isolated from vaginal microflora

Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Khosroushahi AY

J Med Microbiol, 2014 Aug;63(Pt 8):1044-1051.
PMID: 24913559 DOI: 10.1099/jmm.0.074161-0

Forty-five lactic acid bacteria (LAB) were isolated from the vaginal specimens of healthy fertile women, and the identities of the bacteria were confirmed by sequencing of their 16S rDNA genes. Among these bacteria, only four isolates were able to resist and survive in low pH, bile salts and simulated in vitro digestion conditions. Lactococcus lactis 2HL, Enterococcus durans 6HL, Lactobacillus acidophilus 36YL and Lactobacillus plantarum 5BL showed the best resistance to these conditions. These strains were evaluated further to assess their ability to adhere to human intestinal Caco-2 cells. Lactococcus lactis 2HL and E. durans 6HL were the most adherent strains. In vitro tests under neutralized pH proved the antimicrobial activity of both strains. Results revealed that the growth of Escherichia coli O26, Staphylococcus aureus and Shigella flexneri was suppressed by both LAB strains. The antibiotic susceptibility tests showed that these strains were sensitive to all nine antibiotics: vancomycin, tetracycline, ampicillin, penicillin, gentamicin, erythromycin, clindamycin, sulfamethoxazole and chloramphenicol. These data suggest that E. durans 6HL and Lactococcus lactis 2HL could be examined further for their useful properties and could be developed as new probiotics.
Probiotics or antibiotics: future challenges in medicine

Nami Y, Haghshenas B, Abdullah N, Barzegari A, Radiah D, Rosli R, et al.

J Med Microbiol, 2015 Feb;64(Pt 2):137-46.
PMID: 25525206 DOI: 10.1099/jmm.0.078923-0

Genetic and environmental factors can affect the intestinal microbiome and microbial metabolome. Among these environmental factors, the consumption of antibiotics can significantly change the intestinal microbiome of individuals and consequently affect the corresponding metagenome. The term 'probiotics' is related to preventive medicine rather than therapeutic procedures and is, thus, considered the opposite of antibiotics. This review discusses the challenges between these opposing treatments in terms of the following points: (i) antibiotic resistance, the relationship between antibiotic consumption and microbiome diversity reduction, antibiotic effect on the metagenome, and disease associated with antibiotics; and (ii) probiotics as living drugs, probiotic effect on epigenetic alterations, and gut microbiome relevance to hygiene indulgence. The intestinal microbiome is more specific for individuals and may be affected by environmental alterations and the occurrence of diseases.
Isolation and characterization of probiotics from dairies

Haghshenas B, Nami Y, Almasi A, Abdullah N, Radiah D, Rosli R, et al.

Iran J Microbiol, 2017 Aug;9(4):234-243.
PMID: 29238459

Background and Objectives: Probiotics are live microorganisms, which show beneficial health effects on hosts once consumed in sufficient amounts. LAB group can be isolated and characterized from traditional dairy sources. This study aimed at isolating, identifying, and in vitro characterizing (low pH/high bile salt tolerance, antibacterial activity, and antibiotic susceptibility) LAB strains from traditional Iranian dairy products.
Materials and Methods: Isolated strains were identified by Gram staining, catalase assay, and 3 molecular identification methods; namely, (GTG) 5-PCR fingerprinting, ARDRA, and 16S rDNA gene sequencing.
Results: A total of 19 LAB strains belonging to 4 genera (Lactococcus, Leuconostoc, Lactobacillus and Enterococcus) were identified.
Conclusion: The experiments revealed that L. plantarum 15HN, L. lactis subsp. cremoris 44L and E. mundtii 50H strains, which were isolated from shiraz, cheese and shiraz, respectively, displayed a desirable tolerance to low pH and high bile salts, favorable anti-pathogen activity, and acceptable antibiotic susceptibility; hence, they could be considered as novel probiotic candidates and applied in the food industry.
Fulltext Probiotic Assessment of Lactobacillus plantarum 15HN and Enterococcus mundtii 50H Isolated from Traditional Dairies Microbiota

Haghshenas B, Haghshenas M, Nami Y, Yari Khosroushahi A, Abdullah N, Barzegari A, et al.

Adv Pharm Bull, 2016 Mar;6(1):37-47.
PMID: 27123416 DOI: 10.15171/apb.2016.07

PURPOSE: Probiotics are microorganisms, which show beneficial health effects on hosts once consumed in sufficient amounts. Among probiotic bacteria, the bioactive compounds from lactic acid bacteria (LAB) group can be utilized as preservative agents. LAB group can be isolated and characterized from traditional dairy sources. This study aimed to isolate, identify, and biologically characterize probiotic LAB strains from Iranian traditional dairy products.
METHODS: A total of 19 LAB strains were identified by sequencing of their 16S rRNA genes. They were examined for adherence to human intestinal Caco-2 cells and tolerance to low pH/high bile salts and simulated in vitro digestion conditions. Moreover, they were evaluated further to assess their ability to prevent the adhesion of Escherichia coli 026 to the intestinal mucosa, inhibitory functions against pathogens, and sensitivity to conventional antibiotics.
RESULTS: L. plantarum 15HN and E. mundtii 50H strains displayed ≥ 71% survival rates at low pH/high bile salts and ≥ 40% survival rates in digestive conditions. Their adherences to Caco-2 cells were 3.2×105 and 2.6×105 CFU mL-1 respectively and high values of anti-adhesion capability were observed (≥36%). They inhibited the growth of 13 and 11 indicator pathogens respectively. Moreover, they were sensitive or semi-sensitive to seven and three out of eight antibiotics respectively.
CONCLUSION: L. plantarum 15HN and E. mundtii 50H, which were isolated from shiraz product, displayed above-average results for all of the criteria. Therefore, they can be introduced as novel candidate probiotics that could be used in the food industry.
Fulltext Administration of microencapsulated Enterococcus faecium ABRIINW.N7 with fructo-oligosaccharides and fenugreek on the mortality of tilapia challenged with Streptococcus agalactiae

Nami Y, Kahieshesfandiari M, Lornezhad G, Kiani A, Elieh-Ali-Komi D, Jafari M, et al.

Front Vet Sci, 2022;9:938380.
PMID: 35978708 DOI: 10.3389/fvets.2022.938380

We investigated the probiotic potential of a microencapsulated Enterococcus faecium ABRIINW.N7 for control of Streptococcus agalactiae infection in hybrid (Oreochromis niloticus × Oreochromis mossambicus) red tilapia. A two-phase experiment approach was completed in which E. faecium bacteria were propagated, from which a culture was isolated, identified using molecular techniques, and microencapsulated to produce a stable commercial fructooligosaccharide (FOS) and fenugreek (Fk) product of optimal concentration. The FOS and Fk products were assessed in a 90-days in vivo challenge study, in which red hybrid tilapia were allocated to one of five treatments: (1) No Streptococcus agalactiae (Sa) challenge (CON); (2) Sa challenge only (CON+); (3) Sa challenge in a free cell (Free Cell); (4) Sa challenge with 0.8% (w/v) Alginate; (5) Microencapsulated FOS and Fk. In vitro results showed high encapsulation efficiency (≥98.6 ± 0.7%) and acceptable viability of probiotic bacteria within the simulated fish digestive system and high stability of viable cells in all gel formulations (34 < SR% <63). In vivo challenges demonstrated that the FOS and Fk products could be used to control S. agalactiae infection in tilapia fish and represented a novel investigation using microencapsulation E. faecium as a probiotic diet for tilapia fish to control S. agalactiae infection and to lower fish mortality. It is recommended that local herbal gums such as 0.2% Persian gum and 0.4% Fk in combination with 0.8% alginate (Formulation 7) can be used as a suitable scaffold and an ideal matrix for the encapsulation of probiotics. These herbal gums as prebiotics are capable of promoting the growth of probiotic cells in the food environment and digestive tract.

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