In this study, L. plantarum was used as the probiotic bacterium. The inhibitory properties of bacterial metabolites produced by L. plantarum treated with curcumin were then compared to those of metabolites produced by untreated L.

plantarum as the control group. Finally, quantitative real-time PCR was used to examine gene expression changes that contribute to cell survival and apoptosis in order to better understand the mechanisms of action of bioactive metabolites of bacteria against cancer cells.

The CFS of curcumin-treated L. plantarum was utilized to compare its impact with the CSF of intact L. plantarum against HT colon cancer cells. The bacterial culture procedure was repeated for each curcumin concentration until the bacterial populations reached the desired CFU.

In the presence of curcumin, the number of bacterial colonies in de man, rogosa and sharpe MRS agar decreased in the first round of treatment at each concentration but increased in subsequent treatments and adaptation processes. The log CFU for curcumin-treated bacteria was 9.

One of the most distinctive characteristics of all probiotics is their tolerance to low stomach pH, survival rate, and health-promoting properties. In vitro resistance test at low pH was conducted to confirm that curcumin-treated L.

plantarum can survive in the low pH environment of the stomach. The survival rate of bacteria treated with curcumin, as well as their growth at low pH, did not alter considerably Table 1. Probiotics are most well-known for their antibacterial capabilities.

Probiotics, particularly LAB, produce lactic acid, acetic acid, hydrogen peroxide, and bacteriocins which all have antimicrobial properties. The antibacterial capabilities of CFS and cur-CFS were investigated against various pathogen bacteria to confirm that L.

plantarum treatment with curcumin did not impact its probiotic feature. Table 2 summarizes the antibacterial activities of CFS and cur-CFS. The results Fig. The diameter of the zone of inhibition formed by metabolites derived from untreated and curcumin-treated L.

plantarum bacteria against Staphylococcus aureus and Shigella dysenteriae. This investigation was done to investigate the nature of CFS and cur-CFS.

The pretreatment of cur-CFS with proteinase K, eliminates its antibacterial activity, indicating that the secreted proteins from L.

plantarum adapted to the highest dose of curcumin are involved in inducing toxicity against pathogenic bacteria Table 2. The MTT assay was used to determine the cytotoxicity effect of CFS and cur-CFS on HT cells The findings demonstrated that CFS and cur-CFS were capable to induce a cytotoxic effect on HT cells in a dose- and time-dependent manner.

The IC50 value of CFS and cur-CFS were determined as Subsequently, the results indicated that cur-CFS could exhibit more cytotoxic effects on HT in comparison with CFS alone.

The effect of different concentrations of CFS and cur-CFS on the viability of HT cells after 24 h. To evaluate the effect of CFS and cur-CFS on the apoptosis of HT cells, flow cytometric analysis using a FITC-labeled annexin V kit ApoFlowEx FITC Kit was used to identify the early and late phases of apoptosis, as well as the necrosis profiles of the treated cells Exbio, Czech Republic.

The ApoFlowEx FITC Kit is intended for identification of early apoptotic, necrotic and viable cells. Annexin V, a phospholipid-binding protein, binds to phosphatidylserines that translocate to the outer leaflet of the cell membrane during apoptosis.

PI-stained cells only with disrupted cell membranes are quantified to identify late apoptotic and necrotic cells As Fig. Besides, the apoptosis rate was remarkably higher in cur-CFS-treated cells compared to the CFS-treated cells.

Flow cytometry quantification of apoptosis and necrosis in HT colon cancer cells treated with CFS and cur-CFS in comparison with unstained and positive groups.

To determine the effect of CFS and cur-CFS, another flow cytometry analysis was performed in HT cells to assess cell cycle arrest. In the cur-CFS-treated cells, the arrested cells increased by Cell cycle analysis shows the population of cells inters to the Sub-G1 phase in CFS and cur-CFS-treated cells compared to the control group p ˂ 0.

To observe the apoptotic cells in a qualitative manner, the DAPI test was utilized. The nuclei of CFS and cur-CFS-treated HT cells were stained with DAPI to assess the probable chromatin condensation and subsequent DNA fragmentation, which is a hallmark of apoptotic cells.

The shape of the nucleus revealed fragmented chromatin in CFS and cur-CFS-treated cells. The modified shape of the nucleus and fragmented DNA in cur-CFS-treated cells was more than in CFS-treated cells.

However, the DNA of untreated normal cells remained unaltered Fig. Chromatin changes in HT colon cells after 24 h of treatment with metabolites of intact L. plantarum CFS and curcumin-treated L. plantarum cur-CFS. Quantitative PCR Q-PCR was used to measure the amount of gene expression alterations and to further confirm the results of our other tests.

Also, Fig. These results indicate the effect of cur-CFS on the intensification of apoptosis by regulation of this gene. Expression ratios of BAX, Bcl-2, Caspase-9, and Caspase-3 genes in HT colon cancer cells treated with CFS, cur-CFS.

Also, as Fig. These results indicate the effect of cur-CFS on the intensification of apoptosis by regulation of the caspase3 gene.

Cancer is the leading cause of mortality worldwide, making it a serious concern affecting public health on a global scale. The primary cancer treatment approaches are surgery, chemotherapy, immunotherapy, and their combinations Probiotics have recently sparked considerable interest due to their potential for cancer treatment.

Probiotics have been shown to be effective in treating a variety of ailments, including rheumatoid arthritis, psoriasis, autoimmune encephalomyelitis, and gastrointestinal disorders 19 , 20 , 21 , Some of the major health benefits associated with probiotic consumption include the improvement of gastrointestinal microflora, the modulation of the immune system, the reduction of serum cholesterol, the prevention of cancer, the treatment of irritable bowel-associated diarrheas, the effects of antihypertensive medication, and the improvement of lactose metabolism LAB are the most often utilized probiotics as food supplements in this context.

Some LAB strains are thought to boost the immune system, hence preventing colorectal cancer. Oral Acidophilus Lactobacillus enhances host immunity by boosting blood IgG and IgM levels as well as mucus IgA levels.

Furthermore, LAB or their metabolites can interact directly with tumor cells and inhibit their development 24 , 25 , 26 , 27 , The findings of Baldwin et al. indicates that activation of caspase-3 protein was seen following treatment of CRC cells with a mixture of LAB and a chemotherapeutic drug.

They suggested that L. acidophilus accompanied by LAB strain is able to increase the apoptosis induction Haghighi et al. investigated the effects of Streptococcus faecalis , Lactobacillus acidophilus , and Bifidobacterium bifidum on antibody response development. They results revealed that probiotics increase the systemic antibody response to certain antigens in chickens Yurong et al.

showed that probiotics enhanced IgA in the intestinal fluid, IgG-forming cells, IgA-forming cells, and IgM-forming cells in the cecal tonsils of chickens. Their results suggested that probiotics enhance the intestinal mucosal immunity of chickens at the early age Perdigon et al.

investigated the antigenic effect of L. Their results showed that Bcl-2 protein was increased in a dose-dependent manner with all LAB assayed. Their study also demonstrated that LAB strains induce distinct mucosal cytokine profiles It appears that LAB metabolites play an important therapeutic role and may be linked to microbiota and colorectal cancer.

Further investigation revealed that the bacteria possessed a sophisticated mechanism that allowed them to detect nutritional signals and adjust their metabolism accordingly. Our research group previously has revealed that high dosages of propyl gallate PG and tetra butyl hydroquinone TBHQ food preservatives might modify the metabolomics of Lactobacillus rhamnosus and, as a result, have significant anticancer effects when compared to untreated bacteria 5.

plantarum and compare their biological effects with intact bacteria. MTT results demonstrated that CFS and cur-CFS inhibited HT29 cell growth dose-dependently, with a half-maximal inhibitory concentration of DAPI data revealed that cur-CFS-treated cells generated considerably greater chromatin fragmentation within the nucleus than CFS.

Furthermore, flow cytometry investigations of apoptosis and cell cycle validated the DAPI staining and MTT assay results, indicating that cur-CFS-treated cells had a greater incidence of apoptosis about cur-CFS and CFS increased the expression of the Caspase 9,3 and BAX genes while decreasing the expression of the BCL-2 gene, as verified by qPCR.

Dehghani et al. investigated the cytotoxicity, anti-proliferative effects, and apoptotic properties of the supernatant of probiotic Lactobacillus rhamnosus on the HT cell line.

Their findings were in line with our results. Their results showed that the supernatant of L. rhamnosus inhibited the development of HT cancer cells in a dose- and time-dependent manner. Moreover, they affirmed the apoptotic cell death by flow cytometry and qPCR.

We have also observed similar results after pre-treatment of the L. plantarum with curcumin The human colorectal adenocarcinoma cell line HT was purchased from the Pasteur Institute. All of the strains of Lactobacillus plantarum PTCC , Shigella dysenteriyae , Staphylococ aureus , Klebsiella pneumoniae , and Salmonella typhi were bought from the Iranian Research Organization for Science and Technology, Ministry of Science, Technology, and Research Tehran, Iran.

RPMI medium, trypsin, FBS, streptomycin, and penicillin to were supplied by Invitrogen Waltham, MA, USA. Pipettes, tissue culture flasks, and well and 6-well plates were purchased from SPL Life Sciences Gyeonggi-do, South Korea.

Proteinase K, trypan-blue, MTT powder, diethylpyrocarbonate DEPC , Triton-X, and sterile disks were obtained from Sigma Aldrich St. Louis, MI, USA. DMSO, DAPI, curcumin, de Man, Rogosa, and Sharpe MRS agar broth and MRS agar media were provided by Merck Darmstadt, Germany.

RNA isolation kit RNX plus total was bought from Genall. The Reverta-L reagent kit was purchased from InterLabService Moscow, Russia. Power SYBR Green PCR master mix was obtained from Applied Biosystems Foster City, CA, USA.

Propidium iodide PI and annexin V-fluorescein isothiocyanate FITC kits were purchased from EBioscience Waltham, MA, USA. All extra materials, unless otherwise mentioned, were purchased from Merck Darmstadt, Germany or Fermentas Life Science Waltham, MA, USA.

plantarum was cultured in a customized MRS broth medium 37 °C overnight to achieve an optical density OD of 0. MRS agar is developed primarily for the cultivation of lactobacilli from various sources with the intention of producing a defined medium as a substitute for tomato juice agar.

Plate counting on MRS agar found the OD 0. The bacterial density corresponded to the number of bacteria found in the large intestine i.

The supernatants of curcumin-treated bacteria and intact bacteria were adjusted to a pH of 7. The bacterium sediments were washed twice with PBS pH 7.

To undertake cellular studies, both curcumin-treated and untreated bacteria were centrifuged 24 h before treatment and transferred to an RPMI medium. After 24 h, the supernatants were centrifuged to collect the CFS and cur-CFS, and their pH was adjusted to 7.

To test the resistance of curcumin-treated bacteria to the stomach acidic environment, a technique published by was modified and applied The bacteria that were resistant to the highest dose of curcumin were separated and washed with PBS pH 7.

The washed bacteria were immersed in phosphate buffer solution PBS 0. The bacteria's resistance rate was evaluated on MRS agar using the pour plate technique, and the survival of treated and untreated cells was compared. Using equation I , the survival rate of bacteria resistant to the greatest dosage of curcumin was calculated and compared to the intact bacteria:.

where N 1 represents the total viable counts of isolated bacteria in the MRS agar at low pH; and N 0 is the total viable counts of bacteria before cultivation in the acidic media. A disk diffusion test was performed to assess CFS's antibacterial capabilities against clinically pathogenic bacteria.

Gram-negative bacteria Shigella dysenteriyae were used and the gram-positive bacteria included : Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, and Pseudomonas aeruginosa To summarize, all of the bacterial strains were cultivated in nutritional broths.

The plates were then incubated for 24 h at 37 °C before being tested to determine the width of the inhibitory zone. A sterile disk with PBS served as the negative control pH 7. Azithromycin 30 µg and Ampicillin 30 µg were used as positive controls for Pseudomonas aeruginosa, Shigella dysenteriyae, Salmonella typhi, and Staphylococcus aureus Bio-Rad, Hercules, CA, USA.

Tetracycline and cefazolin 30 µg were employed as positive controls for Klebsiella pneumoniae. The antibacterial and cytotoxicity activity of protein-free CSFs were compared to intact CFSs after being inactivated by proteinase K for 20 min at 70 °C The MTT assay was used to analyze the cytotoxic effects of CFSs on HT cells in well plates 34 , Cells were seeded at a density of 2.

The cells were subsequently incubated at 37 °C for 24 h with different dosages of CFSs dissolved in RPMI. Following the incubation durations, the media were removed and µL of MTT reagent 0.

After 4 h of incubation, the formazan crystals formed by the oxidation of the MTT dye in living cells were dissolved in DMSO and the UV absorbance at nm was determined using a Tecan ELISA plate reader Männedorf, Switzerland.

The ApoFlowEx FITC Kit was used to identify the early and late phases of apoptosis, as well as the necrosis profiles of the treated cells Exbio, Czech Republic. After draining the supernatant, the cells were resuspended in µL of BB and 5 µL of annexin V-FITC. The cells were examined using a BD FACSCaliburTM flow cytometer FACSQuant; Milteny, Germany equipped with FITC green emission filters of — nm and PI emission filters of nm red.

Each experiment recorded a total of 10, occurrences. DAPI staining was used to determine the extent of DNA breakage condensed and fragmented DNA generated by various CFSs in the HT cell line using an Olympus IX81 fluorescent microscope Hamburg, Germany 5.

To do this, the cells were seeded at a density of 5. After washing with PBS, the cells were permeabilized with Triton X- 0.

Finally, the images of treated cells were taken using a fluorescent microscope excitation at — nm and emission at — nm. Cell cycle analysis was performed to investigate the apoptotic properties of CFS and cur-CFS. After 24 incubations, the cells were then washed with PBS and 5 μL of RNase A was added to each group and incubated for 30 min at 37 °C.

Afterward, 20 μL PI alongside Triton X 0. The cell cycle arrest was detected with flow cytometry and analyzed by FlowJo software as well. The effect of various CFSs on expression levels of Bax, caspase9, and caspase3 pro-apoptotic gene , and Bcl-2 anti-apoptotic gene was evaluated using quantitative real-time PCR analysis.

The expression level of GAPDH was also investigated as the housekeeping gene. To evaluate the effect of CFS on the expression of specific genes, HT cells were cultured in 6-well plates for 24 h and treated with various CFSs at corresponding IC50 concentrations.

The cells were lysed for RNA extraction after 24 h of incubation at 37 °C A Real-time PCR reaction was performed in a Bio-Rad device. For each gene, reverse and forward primers Table 3 were designed by oligo 7. The qPCR reaction was performed using SYBR® Green master mixes.

Thermal cycling conditions were as follows: 10 min at 94 °C, 40 cycles at 95 °C for 15 s, 30 s at 60—62 °C depending on the gene utilized , and 25 s at 72 °C. The Pfaffl technique was used to examine the data, and the cycle threshold CT values were standardized to the rate of β-actin expression.

Each reaction was performed in triplicate, and each experiment included a negative control. At least three independent tests were done on each set of data collected throughout this investigation.

The mean and standard deviation of continuous variables were used to express them SD. GraphPad Prism version 6. Probiotic bacteria have been shown to have anticancer properties against various types of cancers, including colorectal cancer.

According to research, probiotic bacteria have a specific mechanism that allows them to receive nutritional signals and adjust their metabolism accordingly based on their prebiotics. Previous research has suggested that there is a relationship between nutrition with probiotic biological activities.

Curcumin is a food additive that is popular in many cuisines due to its spicy flavor and vibrant color. Long-term curcumin use may affect the biological characteristics of probiotic bacteria in individuals, particularly their anticancer properties.

Therefore, in this investigation curcumin was used to assess its properties on the antiproliferative properties of the probiotic L. plantarum strain. In this context, L. plantarum bacteria were treated with curcumin and their probiotic features were evaluated in terms of their low pH tolerance potency and antibacterial properties.

Furthermore, the antiproliferative activity of the CFS of curcumin-treated L. plantarum was compared to that of intact L. Curcumin-treated L. plantarum caused more apoptosis against HT colon cancer cells than intact L. plantarum , as demonstrated by cytotoxicity and genotoxicity studies.

The findings in this study suggest that the metabolomics of curcumin-treated probiotics may either directly induce enhanced toxicity against cancer cells or may interact with curcumin indirectly, amplifying their anticancer effects.

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Although only some probiotics currently show anti-cancer properties, the function of other probiotics should be studied. Probiotics as an adjunctive treatment are potentially beneficial in cancer prevention and treatment. It positively affects the metabolism of the gut microbiota during chemotherapy, thereby decreasing its toxic side effects.

Currently, the probiotic treatment of cancer mainly uses combination therapy, including radiotherapy and heat-killing Lactobacillus casei strain LC therapy. Other therapies are waiting to be explored. The research on probiotic inhibition resistance is primarily observed in probiotic inhibition of antibiotic treatment Helicobacter pylori.

However, few studies on probiotics indicate inhibitory resistance in other aspects. Despite this, probiotics still have significant potential in cancer treatment and tumor immunity. ZW, LL, and SW conceived and designed this review. LQ and LP organized the references, prepared figures.

KX wrote and supervised the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

The Science and Technology Research Program of Chongqing Municipal Education Commission Nos. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. SCFAs, Short-chain fatty acids; ND, no data; PBMC: Human peripheral blood mononuclear cells; L.

albicans, Lactobacillus albicans; LGG, Lactobacillus rhamnosus GG; L. plantarum, Lactobacillus plantarum; L. royale, Lactobacillus royale; L.

acidophilus, Lactobacillus acidophilus; LP8, Lactiplantibacillus plantarum CKDB; LPS, lipopolysaccharide; 5-FU, 5-fluorouracil; LBB, Lactobacillus acidophilus, Bifidobacteria bifidum, and Bifidobacteria infantum; DMH, 1,2-dimethylhydrazine dihydrochloride.

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Among them, especially Lactobacillus casei Shirota [LcS] showed a high potential. This hypothesis has elicited further investigations on the anti-tumor and immunoregulatory action of LcS in various experimental models [ 56 — 58 ]. Oral administration of LcS has exhibited beneficial effects in both humans and animals as well as anti-tumor activity against human bladder cancer cells in clinical trials [ 59 , 60 ].

LcS has been shown to possess powerful anti-tumor and anti-metastatic effects on transplantable tumor cells and to suppress chemically-induced carcinogenesis in rodents. In particular, It has been noted that the intrapleural administration of LcS into tumor-bearing mice has induced the production of several cytokines, such as Interferon-γ [IFN-γ], interleukin-β [IL-1β] and tumor necrosis factor-α [TNF-α], leading to the inhibition of tumor growth and to an increased survival [ 58 , 61 ].

Lcs has also exhibited a strong anti-tumor effect in mice by regulating the host immune response in a 3-methylcholanthrene [MC]-induced carcinogenesis model [ 63 ] that has been used to induce many tumors, including colon cancer model [ 64 , 65 ].

An LcS oral feeding of mice is likely to counteract MC-induced tumorigenesis by ameliorating the host immune responses which have been disrupted during MC carcinogenesis. A possible mechanism of carcinogenesis prevention is the proliferation and activation of NK cells [ 66 ].

NK cells are large granular lymphocytes derived from bone marrow, and have a critical role in immune surveillance against tumor development [ 67 ]. Other possible effector cells that may respond to LcS and other probiotics are DCs [ 62 , 68 ]: they represent important types of cells involved in the presentation of several antigens and in the production of cytokines [ 69 ].

In addition, oral administration of LcS has been shown to stimulate type 1 helper T cells, activate the cellular immune system, and inhibit the incidence of tumors and IgE production in mice [ 70 ]. More recently, it has been reported that LcS has suppressed murine tumorigenesis with potent elicitation to produce interleukin [IL] by bone marrow-derived cells in vitro [ 71 ] and to inhibit of interleukin-6 [IL-6] production in the colonic mucosa [ 72 ].

In numerous studies, other probiotic strains have shown remarkable immunoprotective properties through the increase of specific and non-specific mechanisms that have anti-tumor effects. For instance, Lee et al. acidophilus SNUL, L. casei YIT and B. longum HY, for instance, increased the survival rate of mice injected with tumor cells.

These findings suggest that the treatment with probiotics has the potential to prevent CRC by modulation of the host's immune system, specifically cellular immune responses. Apoptosis is a genetically determined mode of cell death playing a key role in the regulation of cell numbers.

In many types of cancer, a reduced ability to trigger apoptosis is an important pathogenetic event that is accompanied by alteration of control processes of cell proliferation [ 74 ].

The regulation of cell survival and death with molecules acting on the apoptotic process can have a huge chemopreventive and therapeutic potential [ 75 ]. There is much evidence that probiotics can have a role in the regulation of cell proliferation and apoptosis which are potentially crucial mechanisms in the prevention of CRC.

Iyer et al. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IkBa ubiquitination and enhancing pro-apoptotic mitogen activated protein kinase [MAPK] signaling. The probiotic mixture VSL 3 has been reported to suppress the COX-2 expression in Colo and SW intestinal epithelial cells [ 77 ].

The expression of COX-2 is increased in colorectal tumors [ 78 ], and this elevation can protect intestinal epithelial cells from apoptosis [ 79 , 80 ]. Recently, rodent studies have demonstrated that the synbiotic combination of resistant starch and Bifidobacterium lactis has exerted a pro-apoptotic action in response to the carcinogen, AOM [ 10 , 81 ].

Other studies have postulated that probiotics possess CRC-protective effects by altering the differentiation process of tumor cells. Using a cultured human colon cancer cell line [HT], Baricault et al. Milks were fermented by one of the following bacterial populations: Lactobacillus helveticus , Bifidobacterium , L.

acidophilus or a mix of Streptococcus thermophilus and Lactobacillus bulgaricus. After HT cells were added to the fermented milk, only L. Concomitantly, the specific activities of dipeptidyl peptidase IV, which is a sensitive and specific marker of HT cell differentiation, and those of three other brush border enzymes [sucrase, aminopeptidase N and alkaline phosphatase] were significantly increased, thus suggesting that these cells may have entered a differentiation process.

This could be explained by the induction from an undifferentiated phenotype to a more differentiated one. In fact, the results showed that cancerous HT cells treated with the synbiotic, when compared with the differentiated ones, reached the same rate expression of intestinal alkaline phosphatase, a biomarker of colic differentiation [ 84 ].

Singh et al. longum resulted in a significant suppression of colon tumor incidence and tumor multiplicity, and it also reduced the tumor volume. Analyses on intermediate biomarkers also revealed that the ingestion of B. longum inhibited AOM-induced cell proliferation through a reduction in ornithine decarboxylase [ODC] activity.

ODC is involved in the biosynthesis of polyamines that cause cell proliferation and differentiation of the colonic mucosa [ 86 ]. According to these data, an improved understanding of LAB-mediated effects on apoptosis and differentiation signalling pathways may facilitate the development of future probiotics-based regimens for the prevention of CRC.

The bacterial transformation of dietary components in the intestinal lumen may be associated with the production of cancer-preventive agents and may therefore be another mechanism whereby probiotics can influence CRC risk.

The bacterial fermentation of indigestible carbohydrates generates short-chain fatty acids [SCFA] and gas; while the gas is eliminated in the feces, SCFA [mainly acetate, propionate and butyrate] represent nutrients and growth signals for the intestinal mucosa and may play a role in CRC prevention [ 87 ].

They reduce, for instance, the concentration of secondary bile salts. Butyrate, that is the most widely studied of these SCFA, is a preferred energy source for colonocytes and is likely to promote a normal phenotype in these cells.

In CRC cell lines, butyrate enhances cellular differentiation and reduces proliferation [ 88 , 89 ]. In human studies, butyrate and the associated lowering of luminal pH are correlated with a reduced risk of CRC [ 90 , 91 ].

A specific strain [MDT-1] of the ruminal bacterium Butyrivibrio fibrisolvens has been evaluated for use as a probiotic to prevent CRC cancer since it produces high amounts of butyrate [ 92 ]. Using a mouse model of colon cancer, the administration of MDT-1 has reduced the number of ACF and the percentage of mice with an increased proportion of ACF.

Furthermore, the human probiotic Propionibacterium spp. has been shown to kill CRC cells through apoptosis in vitro via its metabolites, the SCFA, acetate and propionate [ 93 , 94 ]. However, synbiotics would be more active than probiotics alone in increasing the production of SCFA and consequently protect against CRC onset [ 10 , 95 ].

A possible explanation is that the interaction of the immunomodulating properties of probiotic bacteria and butyrate, which is more produced via fermentation of prebiotics, results in an upregulation of apoptosis [ 10 , 11 ].

In addition to SCFA, probiotics are involved in the production of another group of fatty acids, termed conjugated linoleic acids [CLAs]. These are a group of isomers of linoleic acid that have been shown to exert numerous health benefits, including anti-inflammatory and anti-carcinogenic effects [ 96 , 97 ].

In rodent studies CLA has been shown to reduce the incidence of colonic tumors [ 98 , 99 ]. Using animal models, Ewaschuk et al. These studies support a role for supplemental probiotics as a strategy for preventing CRC by fermentation of indigestible food, but further investigations are needed.

Signaling pathways are represented by a series of biochemical events whereby a cell communicates with the extracellular environment. Signaling pathways are activated by receptors or cytoplasmic proteins with tyrosine kinase activity and play a critical role in carcinogenesis [ ].

Saccharomyces boulardii [ Sb ] is a safety probiotic agent used to prevent or treat a wide variety of human GI disorders [ , ]. It has been reported that Sb acts through modulation of the host signaling pathways that regulate the intestinal mucosal inflammatory response.

In particular, Sb down-regulates MAPK signaling pathways [ , ] that are located downstream of many growth-factor receptors, including the epidermal growth factor receptor [EGFR]. Chen et al. Sb prevented cancer cell colony formation, reduced EGF-mediated cell proliferation, and increased apoptosis.

Both in vitro and in vivo effects were consistent with inhibition of the EGFR and Akt pathways. Furthermore, a laboratory study by Ma et al. Bacillus polyfermenticus exerted its anticancer effect through the reduction of ErbB2 and ErbB3 and their downstream signaling molecules E2F-1 and cyclin D1.

Thus, in addition to the other anti-tumorigenic effects, probiotics may inhibit EGFR and other tyrosine kinase signaling pathways and thereby may also serve a novel therapeutic or prophylactic role in intestinal malignancies. Although a wide range of studies have brought to growing remarkable findings in recent years, it has not still been possible to obtain conclusive clinical evidence supporting the role of probiotics in CRC prophylaxis.

Since CRC is an impractical endpoint in terms of numbers of subjects, cost, study duration and ethical considerations, probiotic intervention studies often use recurrence of preneoplastic lesions or intermediate biomarkers of cancer as an endpoint [ , ].

Several mechanisms could explain the preventive action of probiotics against CRC onset. All of the CRC-preventing mechanisms previously discussed are supported in varying degrees from in vitro and animal model studies, some of them even from human clinical studies.

We are not still able to determine which mechanisms are most effective. Most likely distinct strains of probiotics operate with specific mechanisms. Further investigations are strongly required in order to establish the impact of each mechanism and the real usefulness of probiotics in CRC prevention.

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In recent years, in addition to surgical treatment, the research and development of probiotics has provided novel therapies for the treatment of solid tumors. Probiotics have been shown to have beneficial effects on human health, including antibacterial activity against intestinal pathogens, regulating blood cholesterol levels, reducing the incidence of colitis and inflammation, regulating the immune system, and preventing colon cancer 3.

Most probiotics are found in the natural intestinal flora, and the main component of probiotics is lactic acid bacteria LAB. Lactobacillus and Bifidobacterium are the most common LAB.

Many studies have shown that probiotics can treat a range of gastrointestinal diseases, such as acute diarrhea, antibiotic-associated diarrhea, functional digestive disorders, and inflammatory intestinal diseases, by affecting or even changing the intestinal flora 5.

It can also participate in the dynamic balance and regulation of the intestinal epithelial system. Moreover, probiotics can affect the quality and quantitative composition of intestinal ecosystems 4.

Research has shown that the immunomodulatory and anti-tumor effects of probiotics are particularly remarkable. There has been increasing evidence to support the feasibility of immunotherapy as a promising therapeutic strategy for the treatment of solid tumors 6. One report showed that the appropriate regulation of intestinal microbiota through the intake of specific probiotics may help to prevent tumor formation 7.

plantarum has been shown to inhibit the growth of CT26 colon carcinoma cells in subcutaneous tumor-bearing mice, most likely by changing the tumor microenvironment and promoting the migration of CD8 T cells and natural killer NK cells to the tumor tissue 8.

Another study demonstrated that oral administration of L. casei induced a potent T helper 1 cell immune response and cytotoxic T cell infiltration in the tumor tissue of tumor-bearing mice, resulting in CT26 tumor growth inhibition 9.

This current study explored the inhibitory effects of a probiotic mixture on CT26 cells, both in vitro and in an animal model. Furthermore, the effects of the probiotic mixture on immune regulation were explored. All animals received humane care. Animal experiments were reviewed and approved by the Animal Committee of the First Hospital of Hebei Medical University License number and guidelines for the care and use of animals were followed.

A probiotic mixture was provided by Hebei Inatural Biotech CO. The mixture was composed of B. longum , B. bifidum , L. acidophilus , L. plantarum , resistant dextrin, isomaltooligosaccharides, fructose oligosaccharides, and stachyose. The CT26 cells were evenly seeded into 6-well plates at 1×10 5 cells per well.

Following co-culture for 8 hours, cells were harvested for further follow-up experiments. Cell viability was assessed using the CCK-8 assay Sigma Aldrich, Missouri, USA. At different time intervals of 24, 48, 72, and 96 hours, 10 µL of CCK-8 solution Dojindo, Kumamoto, Japan was added to each well, and the cells were incubated for 1.

The absorbance was then measured using a Promega GloMax Luminescence detector at a wavelength of nm. The absorbance of the CT26 cells in the PM group and the CTL group was compared and used to create a proliferation curve.

For the colony formation assays, CT26 cells from the PM group and the CTL group were inoculated into 10 mm petri dishes 1, cells per dish and cultured for 2 weeks. Colonies were assessed and quantified.

After 24 hours, a single scratch was made in the layer of cells using the tip of a µL fluid transfer gun. Cells were then rinsed with phosphate buffer saline PBS. Photographs of cell migration across the scratched line were recorded at 0 and 48 hours using light microscopy.

Cell migration and invasion assays were performed using membranes with 8 µm pores BD Biosciences, Franklin Lakes, NJ, USA. The chambers were then gently transferred to the well plate, and µL of cell suspension was evenly distributed onto the basement membrane of the chambers and incubated at 37 °C.

Samples were then stained with hematoxylin and eosin violet. Cells on the outer membrane of the chambers were observed under a microscope × magnification , and the number of cells in 10 random fields of view were counted and averaged.

Except for melting of the matrix, all other steps of the migration assay were identical to those of the invasion assay.

To generate the transplanted tumor model, 1×10 6 CT26 cells were injected into the right groin of each mouse. The former group was designated as the CT26 plus probiotic mixture PM group, and the latter was designated as the CT26 control group CTL.

All mice were raised in the same environment with adequate food and water. During the experiment, the survival status of the mice was observed every day.

At day 21, the mice were sacrificed by cervical dislocation. After the mice were sacrificed at the designated time, tissue samples, including the tumor and the spleen, were harvested. The sections were stained with hematoxylin and eosin HE and observed under light microscopy.

Tissue sections were dewaxed and hydrated using a xylene and gradient alcohol series and rinsed with PBS for 5 minutes. The sections were then incubated in methanol or a 0. Subsequently, the tissue was incubated at room temperature with diluted normal serum for 20 minutes where possible, the source of the serum was consistent with the source of the secondary antibody.

After blotting the excess serum, the sections were incubated with a primary antibody CD8, , Abcam, Cambrige, MA, USA for 30 minutes followed by a 5-minute buffer wash. Sections were then incubated for 30 minutes with a diluted biotinylated secondary antibody , Abcam solution and washed for 5 minutes in buffer.

Lastly, the sections were incubated in peroxidase substrate solution until the desired stain intensity had developed. Slides were then rinsed under tap water.

SPSS The measurement data were expressed as mean ± standard deviation. T tests were used for comparisons between two groups and analysis of variance ANOVA was used for comparisons between multiple groups. Cell viability was assessed using the CCK-8 assay kit.

The absorbance of the cells was recorded at four different time points and the levels of cell proliferation were evaluated. These results demonstrated that the probiotic mixture had an effect on the long-term survival of CT26 cells. The protective effect of the probiotic mixture against CTinduced colon carcinoma was evaluated.

On day 1, 1×10 6 CT26 cells were injected into the right groin of each mouse.

It has been shown that Lactobacillus and Bifidobacterium spp. are able to change the expression of genes involved in cell death, apoptosis, metastasis, and cell proliferation by several studies [ 6 ]. Tiptiri-Kourpeti et al demonstrated that Lactobacillus casei ATCC induced apoptosis of colon carcinoma cells by up regulating the tumor necrosis factor-related apoptosis-inducing ligand TRAIL protein which, in turn, decreased tumor incidence in mice [ 5 ].

In addition, Patricia W Lin et al. have confirmed an anti-apoptotic mechanism for the probiotic L. rhamnosus GG on normal IEC cell line [ 32 ].

Moreover, BC had a notable result among other bacterial groups with similar effect as cetuximab In addition, all of the bacterial groups, except L. Since cetuximab and trastuzumab bind to the outer membrane section of EGFR and HER-2 proteins, it is believed that the signaling responses triggered by activation of these two receptors result in apoptosis of cancer cells [ 33 — 35 ].

It might be that the signaling pathways triggered by cetuximab- EGFR binding and BC- cell attachment are equally strong in apoptosis induction. The reason of higher apoptotic effect of bacterial groups compared with trastuzumab could be that they affect multiple signaling pathways concomitantly in addition to HER-2 and EGFR pathways for apoptosis induction which make the total triggered signal stronger than trastuzumab.

In addition, bacteria as living organisms have the ability of quorum sensing their environment, which probably help them synergistically activate stronger apoptotic signals in the cell.

It may prove that a combination of Bifidobacterial strains have a stronger quorum sensing, compared to other tested bacteria, in response to attachment to cancer cells and hence, the higher apoptotic activity of BC compared to other bacterial groups.

In general, the apoptosis rate induced by the bacterial groups does not seem to be merely dependent on the cells intrinsic characteristics e. the number and types of receptors on the cells but also on the characteristics of the bacterial treatments since, for instance, BC had the highest apoptosis rate in LST cells compared to other bacteria, but the lowest rate in IEC cells.

One of these bacterial characteristics could be surface proteins and the attaching power to different receptors which are different among bacteria. In this study, BC had significant EGFR-down regulating effects among LST cells, both at mRNA and protein levels, whilst having the least interruption on IEC cells compared to other treatments in terms of down regulating this onco-marker.

These properties make BC a very efficient anti-EGFR treatment which significantly reduces EGFR in cancer cells whilst having no significant impact regarding EGFR levels on normal cells. LC, on the other hand, had no significant effect on LST in terms of reducing the EGFR expression neither on the mRNA nor the protein levels , but significantly decreased the gene expression in normal cells during h incubation time; therefore, LC seems not efficient in this regard.

There were no preferences between the three bacterial treatments in decreasing EGFR levels. One result to be noted is that all the bacterial groups had significantly less interruptions in the EGFR expression among normal IEC cells, compared to cetuximab and trastuzumab.

Since Lactobacilli and Bifidobacteria are the commensal flora of the gut in many living organisms, it seems logical that they have less interruptions in normal epithelial cells compared to the drugs.

Compared to EGFR, HER-2 mRNA and protein levels was much more efficiently inhibited by the bacterial groups in LST cells. Several receptors and factors on the cell might be affected by these bacteria that all end up influencing the expression of HER-2 in the cell.

LC had a good effect on cancer cells in terms of down regulating HER-2, but it significantly increased the expression of HER-2 among IEC cells as did the drugs.

Therefore, LC might again have anti-HER2 properties of questionable value. The reason why HER-2 expression among IEC cells has unexpectedly increased by the treatment groups in this study remains unclear.

The bacterial groups had a higher effect on down regulation of COX-2 mRNA and protein levels compared to EGFR and, to a lesser extent, to HER BC, with 20 folds decrease in PTGS-2 expression, was again the most effective bacterial treatment in PTGS-2 down regulation among LST cells acted ~ 5 folds more effective than the drugs.

In the meanwhile, BC did not have a notable effect on PTGS -2 expression among normal IEC cells significantly less interruptive than the drugs. For all these reasons, BC was again the most effective treatment, compared to other bacterial groups, in decreasing COX-2 in cancer cells while not having a great impact on this gene among normal cells.

Another interesting remark in this study was that all the bacterial groups, except LC, were stronger in PTGS-2 down regulation among LST cells, compared to cetuximab and trastuzumab.

This might be an expected observation since the PTGS-2 down regulating effects of some probiotic strains have already been reported by several studies [ 16 , 17 ], whilst the main mechanisms of action for cetuximab and trastuzumab are the blockage of EGFR and HER-2 pathways and not the COX-2 expression.

Since these two drugs are used as the comparative measures against potential probiotics for assessing changes in EGFR and HER-2 expressions, it would have been interesting to see whether or not they have any impact on the expression of PTGS-2 gene and COX-2 protein.

Besides, there are studies which have reported an association between the expression of COX-2 with HER-2 [ 15 ] and EGFR [ 37 ], as well with cetuximab resistance [ 38 ].

Cetuximab and trastuzumab were both able to significantly reduce the expression of PTGS-2 gene in LST and also in IEC cells. This might suggest an indirect influence of cetuximab and trastuzumab on COX-2 expression and might affect the signaling pathways in which COX-2 is involved.

The beneficial effects of BC on CRC- prevention was proved by in vivo experiments in this study. BC significantly ameliorated the DAI, almost completely restored the colon length, inhibited the increase in tumor incidence and prevented the progress of tumors to higher stages and grades.

Studies by Le Leu et al. showed that the probiotic Bifidobacterium lactis synbiotically combined with resistant starch significantly prevented CRC among rat-AOM model as well as facilitating apoptotic deletion of carcinogen-damaged cells in rat colon [ 4 , 7 ].

In another study, Challa et al. demonstrated that the probiotic Bifidobacterium longum combined with lactulose significantly suppressed AOM-induces colonic crypt foci which are preneoplastic markers [ 3 ].

Overall, considering the effects on both the cancer and normal cell lines, Bifidobacteria cocktail is the most efficient treatment, compared to other bacterial combinations used in this study.

In general, this potential probiotic could be considered as a suitable nutritional supplement to be used along with the drugs cetuximab and trastuzumab to treat and prevent CRC. Since the results of this study is probably strain- and cell type- specific, it is recommended that more variants of Bifidobacterial strains and cell types be investigated to obtain a more comprehensive conclusion on the anti-CRC mechanisms of action of this bacterium.

Effects of the bacterial groups on LS T cancer cells A ; cetuximab and trustuzumab drugs on LS T cancer cells B ; bacterial groups on IEC primary cells C ; cetuximab and trustuzumab drugs on IEC primary cells. The authors would like to thank the team in the epidemiology and virology departments of Pasture Institute of Iran especially Dr.

Arash Arashkia, Mr. Mohammad Sadegh Shams, and Dr. Mina Bahrololumi for their kind assistance with this project.

Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Corrections 19 Nov Asadollahi P, Ghanavati R, Rohani M, Razavi S, Esghaei M, et al. Abstract Introduction Probiotics are suggested to prevent colorectal cancer CRC.

Materials and methods Anticancer effects of potential probiotic groups were investigated following of in LST cancer cells compared to IEC normal cells. Conclusions BC was the most efficient treatment in this study.

Pizzo, Duke University School of Medicine, UNITED STATES Received: January 16, ; Accepted: April 25, ; Published: May 13, Copyright: © Parisa et al. Introduction Colorectal cancer CRC is the third most common type of cancer, being surpassed by only lung and breast cancers, and the second cause of cancer-related deaths worldwide [ 1 ].

Materials and methods Treatment groups The anti-cancer effects of the 5 following treatment groups were investigated: 1. Cell lines The anticancer properties of the bacterial groups were assessed on 2 cell lines including the human colon adenocarcinoma cell line LST and the rat normal non-transformed intestinal cell line IEC [ 24 ] as the control cell line.

Cetuximab and trastuzumab anticancer drugs study controls and drug treatment on cells The anti-EGFR and HER-2 monoclonal antibodies cetuximab Erbitux; Merck, Germany and trastuzumab Herceptin; Aryogen Pharmad, Iran were used as controls in this study. Quantitative real time PCR of the genes EGFR , HER-2 and PTGS-2 The expression levels of EGFR , HER-2 and PTGS-2 genes, as onco-markers, were assessed using real time PCR.

Download: PPT. Table 1. Primer sequences used for the real time PCR of the genes EGFR , HER-2 and PTGS Western blotting of the proteins EGFR, HER-2 and COX-2 The expression levels of the onco-marker proteins EGFR, HER-2 and COX-2 were investigated using Western blot.

Animals and bacterial treatments The anticancer properties of the most effective bacterial treatment in in vitro experiment BC were further investigated in vivo.

Ethics All animal experiments were approved by the Animal Care and Research Advisory Committee of Iran University of Medical Sciences ethical code: IR. Tumor assessment and tissue preparation Sixty four days after AOM injection, all mice were sacrificed by rapid cervical dislocation, excised and opened longitudinally to remove their colons.

Statistical analysis Data were expressed as mean ± SD. Results MTT anti-proliferative assay The MTT assay was used as an anti-proliferative assay to find the best treatment dose for the bacterial and drug groups for treating the cell lines.

Apoptosis assessment Apoptosis rate was assessed by flow cytometry as a metric of evaluating anticancer properties of the bacterial groups in this study. Effect on LST cells.

Fig 1. Quantitative real time PCR of the genes EGFR , HER-2 and PTGS-2 Real time PCR was used to assess the expression levels of EGFR , HER-2 and PTGS-2 genes, as onco-markers in colorectal cancer. EGFR expression among LST cells. Fig 2. Relative fold change relative to untreated control cells of the EGFR gene among LST and IEC cells.

EGFR expression among IEC cells. HER-2 expression among LST cells. Fig 3. Relative fold change relative to untreated control cells of the HER-2 gene among LST and IEC cells.

HER-2 expression among IEC cells. PTGS-2 expression among LST cells. Fig 4. Relative fold change relative to untreated control cells of the PTGS-2 gene among LST and IEC cells. PTGS-2 expression among IEC cells. Cetuximab and trastuzimab both decreased PTGS-2 expression by 4.

Western blotting of the proteins EGFR, HER-2 and COX-2 Western blot analysis was performed to evaluate the expression levels of the proteins EGFR, HER-2 and COX Fig 5. Western blot analysis demonstrating the expression of EGFR, HER-2 and COX-2 proteins in LST and IEC cells. Animal treatments BC was the most effective treatment in vitro , and therefore its anticancer properties were further investigated in mice models.

Fig 6. Macroscopic and histological assessment of colic tumor and inflammation in mice. All these results confirm the preventive role of BC in CRC occurrence among CRC animal models. Conclusions Overall, considering the effects on both the cancer and normal cell lines, Bifidobacteria cocktail is the most efficient treatment, compared to other bacterial combinations used in this study.

Supporting information. S1 Fig. s TIF. S2 Fig. Flow cytometry analysis of cancer LST and normal IEC cells before and after treatment with potential probiotics and drugs. s PDF. S1 Table. The relative expression of EGFR, HER-2 and COX-2 proteins among LST and IEC cells.

S1 Raw images. Acknowledgments The authors would like to thank the team in the epidemiology and virology departments of Pasture Institute of Iran especially Dr.

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In a study carried out by Stidl et al. longum , L. acidophilus , L. bulgaricus , L. casei , L. helveticus , L. kefir , L. plantarum and S. thermophiles either contained in fermented foods or in the human gastrointestinal tract were explored regarding their binding capacities to five HCA including AαC, PhIP, IQ, MeIQx and DiMeIQx.

Among the tested eight species of Lactobacillus, L. helveticus and S. thermophilus were seven to eight times more effective than L. Kefir and L. plantarum strains in detoxification.

Faridnia et al. longum BB, and E. coli ATCC to heterocyclic aromatic amines, including Trp-p-2, IQ, MeIQx, 7,8DiMeIQx and PhIP at pH 5. The effect of bacterial cell concentration on binding ability was also monitored.

Results indicated that B. pseudocatenulatum G4 was the most effective strain in binding to HCA compounds followed by B. longum , and E.

It was concluded that gram-positive bacterium, due to its cell wall structure, was more efficient compared with gram-negative strains. Binding to mutagens was pH dependent and the maximum binding ability were observed at pH 6.

The interaction between HCA compounds and various concentrations of B. The impact of pH on binding capacity has been illustrated in previous studies 38 , 45 , 65 , In rats, absorption of HAAs in stomach occurred at pH above 4 Binding of HAAs to L.

acidophilus and B. Contrary to these results, Tsuda et al. reported a maximum binding of Trp-P-1 to L. plantarum mutant strain at pH 8 Detoxifying effect of L.

It was implied that none of the HCA compounds affected the growth and survival of L. casei DN during 24 hours and hours incubation in MRS broth and modified MRS broth, respectively.

In modified MRS broth, decreasing HCA concentration was lower as a result of lower cell density and was dependent on the growth phase of bacteria. The antigenotoxic capability of the probiotic L. rhamnosus IMC was investigated in mice treated with PhIP.

It was also reported that the extent of DNA damage in colon cells significantly decreased, whereas no genotoxic effect was recognized in liver cells casei LOCK , and L. paracasei The same pattern was observed in the decline of β-glucosidase activity.

casei and in the adult group, there was no significant change in the activity of the enzymes. The greatest inhibition extent It is assumed that probiotics can adhere to colonocytes and restrict absorption of mutagens to the intestine 45 or decrease their bioavailability In another study by the aforementioned author, the impact of the same probiotic strains was evaluated on the fecal enzyme and genotoxic activity in human fecal water children, adults and the elderly in the presence of PhIP.

In terms of β-glucosidase activity, probiotic strains induced a slight change in children and adults. paracasei were the most effective in decreasing genotoxicity in children and adults.

In the elderly group, L. casei was the most efficient strains In another study by Klewicka et al. the protective effect of feeding beetroot juice fermented with Lactobacillus brevis and L.

paracasei against aberrant crypt foci ACF formation in rat colon in the presence of PhIP. Lactofermented beetroot juice decreased the number of ACF from 59 to 26, malondialdehyde in the liver and cytotoxic and genotoxic effects of fecal water in PhIP- treated rats In a study by Tavan et al.

a mixture of three HCA- IQ, MeIQ and PhIP were given to male rats for a 7 week period with a cumulative dose of mg of the HCA per kg body weight. thermophilus DN fermented milk on aberrant crypt foci ACF induction initiated by HCA compounds were determined.

It was stated that consumption of milk, especially fermented milk significantly abolished the number of ACFs in rats.

thermophilus fermented milk. Additionally, HCA metabolism, fecal mutagenicity and colon DNA lesions were declined and there was no difference between two strains in terms of protective effect In a related work by Duangjitcharoen et al.

plantarum were determined. At the beginning of the test 0 minute , sudden binding of all strains to mutagen compounds was observed. The highest binding percentage to PhIP plantarum CM4. It was deduced that this new strain could be utilized for human consumption as a protective agent against undesirable compounds in food and reactive metabolites in the gastrointestinal tract Degradation of 16 different PAH compounds 0.

bifidium , S. thermophilus and L. bulgaricus was studied in MRS medium during different incubation periods 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours at 37°C. bulgaricus was prepared. It was claimed that PAH decline was related to bacterial species and incubation period.

bulgaricus , respectively. It is noteworthy that the maximum reduction of PAHs by B. bifidium and S thermophilus was observed after incubation for 10 and 12 hours, and was found to be However, the highest reduction by L. bulgaricus was recorded after 48 hours and was found to be At the end of incubation time 72 hours , the PAHs reducing capability was as follows: L.

bulgaricus thermophilus bifidium The results of PAHs biodegradation in yogurt showed an inconsiderable reduction within incubation periods 1, 2 and 3 hours and the reduction percentage was 3. It was assumed that the existence of PAHs depend on a number of factors such as the type of microorganism, the interaction between microorganisms, the microbial concentration, the composition of the medium, and the microbial growth conditions of temperature and pH The binding ability of lactic acid bacteria isolated from rice and wheat miso to eight different HCA was investigated.

The experiment illustrated that all of the bacterial isolates could bind to Trp-P-1, Trp-P-2, MeAaC, and PhIP efficiently. Hence, bacterial and mutagen types were the factors affecting the binding proportion It was assumed that Van der Waals hydrophobic interactions were important factors in the binding of mutagens.

It was stated that more hydrophobic compounds like AαC and DiMeIQx are bound more efficiently than IQ and PhIP.

Furthermore, the tryptophan pyrolysates are more hydrophobic than the quinolines, quinoxalines and PhIP and are removed better than other HAA compounds For subsequent studies, two isolates were selected which were distinguished as Pediococcus. acidilactici and named as P. acidilactici 1 and P. acidilactici 2.

In the next step, cell wall fractions, heat-treated cells, and cytoplasmic contents were evaluated for their binding ability to HCA compounds.

It was shown that except cytoplasmic content, pure cell wall and peptidoglycan fraction in both strains possessed more binding capability in comparison to bacterial cells. Heat treatment of lyophilized cells of both strains did not modify binding capacity and therefore, binding of the mutagens by cells is not the mechanism involved.

Also, enzymic treatment with various enzymes had no impact on binding except a decrease in enzyme activity. The authors inferred that binding activity of the cell walls of bacteria and cells as a whole were not influenced by the damage; therefore, extracellular substances or structures had no function in this procedure.

When HCA compounds were acetylated, none of the two strains were able to bind the mutagens which were attributed to substitution of the amino group by the acetyl group and indicating the role of the amino group in the binding property. The proposed mechanism of binding activity was the reaction of peptidoglycan with amino group of mutagen compounds In a study by Zsivkovits et al.

the effect of four Lactobacillus strains consisting of L. bulgaricus , S. thermophilus F4, S. thermophilus V3 and B. longum BB on DNA damage induced by HCAs, which are generally found in fried beef beef mix and chicken mix, in the liver and colon of female rats were examined.

Lactic acid bacteria were either administered simultaneously or at different time intervals before giving HCA. Hence, consumption of probiotic dairy products several hours before cooked and fried meats would be beneficial considering the reduction of DNA damage Terahara et al.

surveyed absorption of Trp-P-1 and MeIQx by L. delbrueckii ssp. bulgaricus and S. thermophilus in distilled water, buffer solutions and intestine.

They pointed out that the amount of bound Trp-P-1 and MeIQx in strain was In addition to these findings, it was specified that the absorption of the mutagens was pH dependent. The highest binding of strain to Trp-P-1 happened at the range of but strain bounded to Trp-P-1 and MeIQx at pH 7.

The results of HCA absorption in the small intestine of rats by loop test showed that strain was effective in the binding reduction of Trp-P-1than strain This was pertained to the similarity of the pH of absorption of Trp-P-1 by strain is similar and the small intestine 6 , 7 , It was proved that incorporation of lyophilized cultures of B.

longum BB 0. In a study potential binding ability of the goat probiotics L. reuteri DDL 19, L. alimentarius DDL 48, Enterococcus faecium DDE 39 and B. Also, the B[a] P- probiotic complex was stable after washing with DMSO In April , the Swedish Food Administration found a remarkable amount of acrylamide in various heat treated carbohydrate-rich foods such as potato chips and crisps, coffee and bread 75 and thereafter it was classified as a probable human carcinogen by the International agency for research on cancer Acrylamide is an electrophile molecule and thus can react to nucleophilic groups such as amines, carboxylates, and those that are commonly found in biological molecules such as DNA.

Exposure to acrylamide causes DNA damage and at high doses, neurotoxic and reproductive effects have been observed while exposure to low, but prolonged doses, results in peripheral neuropathy with the presence or absence of central nervous system complications 76 , Because of the undesirable impacts of acrylamide on human health, many strategies have been investigated in order to alleviate the amount of acrylamide in foods.

These approaches include reduction of precursors in raw materials 78 - 80 , changing the process parameters such as temperature, pH and addition of amino acid and salts 81 - 84 and post processing approaches like chromatography, evaporation, polymerization 85 - Recently, application of specific strains of lactic acid bacteria has been explored owing to their ability to reduce the acrylamide content in foods.

Serrano-Nino et al. evaluated the potential ability of 14 lactic acid strains L. casei Shirota SHI , L. reuteri northern regional research laboratory NRRL LR , L. johnsonii ATCC JH , L. acidophilus ATCC AC , L. fermentum ATCC FER , L. rhamnosus ATCC RHA , L. helveticus ATCC HL , L.

casei ATCC L , L. casei L9 L9 , L. casei L30 L30 , L. casei 12A 12A , L. casei 7R1 7R1 , L. Stability of bacterial- acrylamide complex was also determined. It was implicated that the acrylamide binding abilities were pH, concentration, and strain dependent.

Binding to acrylamide varied with respect to incubation time. At 0 hour, the amount of bound acrylamide was from It was proposed that binding was a rapid process and occurred passively on the bacterial surface After 4 hours, L had the best binding ability Generally, Strains JH, FER and L displayed the weakest ability to bind AA, whereas LR strain was the best one at any of the incubation period time.

By increasing the time from 0 to 4 and 12 hours, the amount of AA bound by strains JH, SHI, L 30, 12A, DCP and 7R1 enhanced notably. The binding ability of strains was investigated at different pH levels 3, 5 and 8. The maximum binding at pH 8 was observed in the strain L while at pH 3, a substantial reduction in binding ability occurred Similarly, Zhang et al.

that implied, L. reuteri NRRL and L. casei Shirota bound more effectively at pH 7 than pH 8 It was announced that the probable mechanism of pH influence on binding ability was due to competition between toxic compounds and protons to attach to the negatively charged binding sites The Bacterial-toxin complex was not degraded after three washes with PBS solution which demonstrated that acrylamide binds to the strains irreversibly.

Finally, it was concluded that all the strains had the ability to bind acrylamide at different incubation periods and can be a new detoxification tool for improving the amount of acrylamide.

In another attempt by Serrano-Niño et al, the interaction of acrylamide and aflatoxin B1 with teichoic acids TA in the cell wall of the aforementioned lactic acid bacteria was studied.

TA was extracted from the cell wall and in order to analyze its components, it was subjected to acid hydrolysis.

TA was composed of ribitol, glycerol, glucose, D-alanine and phosphate. The results of binding assay at 0, 4 and 12 hours incubation at 37°C revealed that binding of acrylamide was relevant to strain type and incubation time. The maximum binding at 0, 4 and 12 h was detected in the strain L.

As it was illustrated before, the bacterial-toxin complex was stable and no detectable amount of acrylamide was liberated after three washes. The mechanism of physical binding of toxins to lactic acid bacteria was explicated.

It was proposed that there was a relation between components of TA and percentage of bound acrylamide. The presence of the lower amount of glucose, D-alanine or teichoic acid caused more binding of acrylamide to the cell wall of bacteria.

H-bonds may develop between carbonyl oxygen and the amino group between adjacent acrylamide and D-Alanine directly attached to position D-4 L-2 of ribitol Furthermore, the amine group of D-alanine might react with acrylamide units by means of a Michael addition reaction Concerns about the occurrence of N-nitroso compounds NOCs in foods is growing, since these compounds can induce tumor growth in human and IARC has classified a number of nitrosamines as probably Group 2A or possibly Group 2B carcinogenic to humans They are formed by the interaction of secondary or tertiary amines with proper nitrosating species and their existence in food is a consequence of various processes during production, storage, cooking and in some circumstances through migration from packaging materials The ways to mitigate nitrosamine formation in food include reducing nitrite level in curing salt, application of nitrite substituent, using ascorbic acid as an inhibitor agent, utilization of lower temperatures and indirect heating There are also few studies concerning the inhibitory effects of probiotics which can bind to these compounds.

Antimutagenic activity of some lactic acid bacteria from fermented milk was assessed against N-nitroso-diethylamine NDEA , N-nitroso-dimethylamine NDMA , N-nitroso-piperidine NPIP and N-nitroso-pyrrolidine NPYR.

Among the tested bacteria, the highest inhibitory activity was observed in genus Leuconostoc. The strains consisting Streptococcus lactis ssp.

diacetylactis R, Streptococcus cremoris R, and Leuconostoc paramesenteroides R and R-8 depicted the most inhibitory activity versus the mutagenicity of NDEA. Therefore, these four strains were selected to evaluate their impact on mutagenecities of NDMA, NPYR, and NPIP.

Mutagenecity of NDMA was partly inhibited by the lactic acid bacteria tested and in the case of NPYR and NPIP, these four strains were not so effective. The inhibitory effect of filtrates of cell suspension of lactic acid bacteria on NDEA mutagenicity was also investigated and a strong antimutagenic activity was observed In a study by Grill et al, the influence of NDMA, NPIP and NPYR on growth of six bifidobacteria strain B.

breve ATCC , B. infantis ATCC , B. longum ATCC , B. longum BB and B. animalis ATCC during 24 hours in TYP medium was studied.

longum BB was able to metabolize nitrosamines. The inhibitory effect of bifidobacteria was attributed to an intracellular enzymic activity Nowak et al studied binding and degrading ability of five probiotic Lactobacillus strains L. rhamnosus LOCK , L.

casei LOCK, L. casei DN and L.