IMPORTANCE OF PROBIOTICS IN GASTROINTESTINAL TRACT

Kiran Konain¹ --- Sadia² --- Turfa Nadeem³⁺ --- Adeed Khan⁴ --- Warda Iqbal⁵ --- Arsalan⁶ Amir Javed⁷ --- Ruby Khan⁸ --- Kainat Jamil⁹ --- Saleha Qazi¹⁰

¹Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan
^2,4,5,8,9,10Centre of Biotechnology & Microbiology, University of Peshawar, Peshawar, Pakistan
³Department of Pharmacy, The University of Lahore, Islamabad Campus, Pakistan
^6,7Institute of Biotechnology & Genetic Engineering, The University of Agriculture, Peshawar, Pakistan.

Keywords:Probiotics, Gastrointestinal infection (GIT), Microflora, Microbial equilibrium, Clinical importance, Adverse effects

ARTICLE HISTORY: Received: 16 March 2018, Revised: 9 April 2018, Accepted: 13 April 2018, Published: 16 April 2018

Contribution/ Originality:This study is one of very few studies conducted in Peshawar on probiotics and can be used as baseline information for further research in Pakistan. The paper's primary contribution is finding the role of probiotic in GIT infection and documents how it affect gut micro-biota and its utilization as effective treatment option.

1. INTRODUCTION

Gastrointestinal tract (GIT) infection is one of the significant reasons for deaths around the globe due to severe dehydration and diarrhea. The decease rate of children under age of five is about 1,575,000 from the inspection done in 2006 [1]. Micro-organisms live in every habitat including the human body in the mouth, gastrointestinal tract and even on the skin. These microorganisms have such an association in which one organism depends on another organism for nourishment or different advantages without harming or helping it. Within the human body, higher concentrations of these commensals micro-organisms are found in the gastro-intestinal tract. GIT has more than 500 different species of bacteria. Among them, some have a positive effect on human health [2]. It has been assessed that microscopic organisms represent 35-50% human colon volume and comprises of Clostridium, Bifidobacterium, Lactobacillus, Bacteroides, Fusobacterium, Peptococcus, Eubacterium, Escherichia, Veillonella and Peptostreptococcus [3, 4].

1.1. Influence of Probiotics on Humans and Animals Health

A specialist board authorized by WHO and Food and Agriculture Organization (FAO)  characterized probiotic as "live micro-organisms," which, when given in satisfactory sums gives a medical advantage to the host [5]. Probiotics can influence both human and animal health by modulating microbiota of the intestine at present, food ingredients of the particular live microbes and their impacts on human wellbeing are concentrated on both inside of food matrices. Furthermore, as single or blended culture preparation [6, 7]. and micro-organisms should have following properties for them to be used as probiotics: i) it should be safe to use; ii) it should be able to resist the action of hydrochloric acid, bile and pancreatic juices; iii) it should not be carcinogenic; iv) it should have the ability to survive in both in acidic as well as  basic environment of stomach and duodenum respectively [8] v) should have positive effect on host; vi) should not be pathogenic and toxic [9] vii) should be able to stimulate immune system and can produce lactic acid. Foods that mainly contain lactic acid bacteria include cheese, wine, fermented milk, sausages and fruit juices [8].

Table-1. Criteria of an ideal microorganism as probiotics [10]

1: should be safe to use

2: withstand the action of hydrochloric acid, bile and pancreatic juices

3: should be non-carcinogenic and non-pathogenic

4: should be able to persist both in the acidic and basic environment of stomach and duodenum

5: should effect host positively

6: can stimulate the immune system

7: can produce lactic acid

Adapted from Singhi and Baranwal [10]

1.2. Characteristics of Probiotics

1.2.1. Mode of action

The consumption of probiotics has several positive effects such as by the regulation of microbiota it can enhance the intestinal tract health, immune system stimulation, augment the bioavailability of nutrients, bringing down the risk of some maladies and reduction in the signs related to intolerance of lactose. Different mode of actions has been suggested that describe the positive effect of using probiotics such as altering the pH of gut, completion with the pathogen for binding receptor sites, producing anti-microbial compounds, production of lactase and stimulation of immunomodulatory cells. However, the specific mechanism used by probiotics to influence their effects is still unknown [11]. Different strains of bacteria differ on the basis of their effect on health. Keeping in mind the possible targets of probiotic, they are designed and applied to specific diseases. The interaction of host with microbes can be of the following type: i) interaction of microbe-gut epithelium; ii) interaction between microbe-immune system; iii) microbe-microbe interaction.

1.3. Adhesion

Adhering to the mucus of intestine is a basic requirement for the colonization and is important in the modulation of the immune system [12-14]. Various investigations uncover that probiotics can competitively restrain the pathogen attachment like that of Staphylococcus aureus, Escherichia coli, Clostridium difficile, Salmonella, Listeria monocytogenes, and Bacteroides vulgatus [15-20]. It is also reported that some Bifidobacteria and lactobacilli share carbohydrate binding specificities with some of the enteropathogens [21]. It is additionally revealed that some Bifidobacteria and lactobacilli impart starch restricting specificities to a portion of the enteropathogens.  For the most part, it is trusted that the strains of probiotics can repress pathogenic bacteria adhesion by steric hindrance at intestinal absorptive cells pathogen receptors. Thus, the main basis of selection of new probiotic strain is adhesion.

1.4. Anti-Microbial Substances

The metabolites that are manufactured by lactic acid bacteria having antimicrobial effects can be partitioned into two sets (i) bacteriocins that are antimicrobial proteins (>1000Da) and (ii) compounds with low molecular mass (below 1000Da) [22, 23].

Probiotics produce acids from the fermentative metabolism of carbohydrates are considered as the main antimicrobial compounds which are accountable for their inhibitory effect against pathogens [24, 25]. Probiotics also show antagonistic effects against different pathogens belonging to genus Salmonella, Escherichia, Listeria, Shigella, Clostridium, Candida, Helicobacter and Campylobacter [26-30].

1.5. Immunomodulation

Studies show that use of probiotics can repair and prevent damage to the intestine. Probiotics can regulate and stimulate natural as well as acquired immune response by interaction with mucous associated lymphoid tissue. This interaction has a high importance for human wellbeing and could affect infectious diseases such as allergic diseases, some types of cancer, a variety of intestinal inflammatory diseases and auto immune disorders. Probiotics bind with the receptors present on the epithelial cell surface, inducing cellular and humoral immune response that direct to both pro-inflammatory and anti-inflammatory responses. There are expanding evidence that a couple of probiotics can produce defensive immune response enough to raise resistance to microbial pathogens [31]. Studies done on human and animal shows that different probiotic strains can affect immune system differently. These effects also depend on the immune status of the host as well as the dose given [32].

1.6. Typical Human Gut Micro-Flora as a Source of Probiotics

The human intestine is accompanied by trillions of microbes that form a complex community influencing the sensitivity towards diseases and normal physiology by a complex interaction between host and the microbes [33]. Although these microbes are present all through the intestine, the high concentration and metabolic activity of these are present in the large intestine [34, 35]. Data obtained from cultures shows that complex microflora present in mouth consists of facultative anaerobes which can include Bacterioides, Yeasts, Streptococci and Lactobacilli. The upper portion of bowel has scanty micro-flora up to 105 colony forming units/ml. From ileum and onward, the concentration of these bacteria’s increases slowly and reaches up to 1011 to 1012 colony forming units/g within the colon. Approximately 500 various species of microbes exist, although on a quantitative basis 10-20 genera might predominate in healthy human microflora including Clostridium, Bifidobacterium, Bacterioides, Peptococcus, Lactobacillus, Veillonella, Peptostreptococcus, Fusobacterium, Escherichia and Eubacterium [34]. Any change in the composition of gut microbes can lead to diseases like gastric ulcer, colon cancer, auto-immune disease, cardiovascular diseases, diabetes type 2 and obesity [36]. Probiotics can have an immediate antimicrobial impact. Truth be told, a few strains/species of probiotics can specifically inhibit or kill the development of pathogenic microscopic organisms like bacteria through creation of antimicrobial components, for example, bacteriocins, proteases coordinated against bacterial toxins, or through avoidance of pathogens by basically sticking to epithelial cells [37-39].

2. CLINICAL IMPORTANCE OF PROBIOTICS

2.1. Diarrhea Associated with Antibiotics

About ≤ 20% of patients suffer from diarrhea after taking antibiotics. This is due to the imbalance in microbes that can cause decrease in the flora, the result is resistance to colonization and decrease in the capacity of fermentation of the colon [40]. Even though new antibiotics that are effective against a large verity of micro-organisms with lesser side effects, have been developed, still antibiotic associated diarrhea ranges from 3.2-29/100 patients [41]. Antibiotics that contain anaerobic bacteria (penicillin, cephalosporin and clindamycin) are associated with more antibiotic associated diarrhea rates. Several meta-analysis reports published recently showed that use of probiotics decreased the occurrence of AAD [42-45]. Mostly used probiotics are lactic acid bacteria, S. boulardii and many combinations of LABs administer in 107 to 1011 doses with duration of 5-49 days parallel to the duration of an antibiotic therapeutic procedure. One of the meta-analyses showed that L. rhamnosus, S. boulardii and many mixes of two dissimilar probiotics are most effective against AAD [45].

2.2. Diarrhea Linked with Infections

Use of probiotics can reduce the duration of infectious diarrhea in both children and adults. From different trials, it was shown that there was about 30 hours a decrease in symptoms durations. From Cochrane review involving 23 studies with about 2000 participants out of which 352 were adults, it was deduced that use of probiotics reduces persistent diarrhea risk. Only two studies used S. boulardii while mostly tested probiotics (18 out of 23 studies) were LABs [46]. The effectiveness of probiotics in treating infectious diarrhea has also been evaluated in a meta-analysis which included total of 63 studies out of which 56 were infant/young children. Altogether, probiotics reduced the duration of diarrhea by 24.76 h (95% CI 15.9–33.6 h), the risk for diarrhea lasting ≥4 days (risk ratio 0.41; 95% CI 0.32–0.53), and reduced stool frequency on day 2 (mean difference 0.80; 95% CI 0.45–1.14) [47].

2.3. Travelers’ Diarrhea

A typical health problem among travelers is traveler’s diarrhea. Rates of this diarrhea can extend from 5 to 50% contingent on destination. A meta-investigation was done on distributed randomized controlled clinical trials of cases involving traveler’s diarrhea. It was reasoned that probiotics fundamentally avert the occurrence of traveler’s diarrhea. Saccharomyces boulardii and a blend of Bifidobacterium bifidum and Lactobacillus acidophilus had noteworthy efficacy [48]. Acute diarrhea occurs to about half of travelers who visit high-risk areas. Although many cases are mild and self-limiting, there is a considerable morbidity [49, 50]. Different studies were conducted with the use of probiotics. Few studies involved lactobacilli which showed negative results, however, 4 studies used a variety of probiotics that showed positive results. 94 Danish tourists who participated in a trip of 2 weeks to Egypt were treated with a mixture of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacteria, and S. thermophilus or a placebo in a randomized study. The rate of traveler's diarrhea was decreased from 71% (very high) to 43% [51].

2.4. Inflammatory Bowel Disease

Inflammatory bowel diseases are characterized by recurrent or chronic intestinal infections with an unknown cause. These disorders include chronic disease, ulcerative colitis, and pouchitis. The process for maintenance and initiation of this inflammatory process has not been discovered, but main theory stated that inflammatory bowel disease results from an abnormal response of the host to some floral members of an intestine or from mucosal barrier defect [52, 53]. Different studies show the effect of probiotics on inflammatory bowel disease in animals. The administration of L. reuteri in rats prevent the formation of acid induced colitis [54]. In Crohn's disease, oral bacteriotherapy with L. casei strain GG has shown same effects. In disease of crowns, there is an increasing number of IgG together with mucosal IgG deficiency in lesions of tissue. Oral application for ten days of L. casei strain GG led to an increase in IgA mucosal levels. Results suggest immunological barrier improvement [55]. In open studies 10-d administration of L. rhamnosus GG with the 14 children having inactive or active crohn disease results in increase immunoglobulin secreting cells to casein and beta-lactoglobulin indicating interaction within the immune system and probiotic [55]. Hotz and Plein conducted a double-blinds, plot, controlled study of S. boulardii of efficacy on Crohn Disease Symptoms. 20 patients with moderate, active Crohn's disease were assigned randomly to a placebo for 7 weeks or S. boulardii to a standard treatment. The significant decrease in frequency of disease activity and bowel movement was observed in the having S. boulardii and not in the placebo group [56]. The efficacy of oral E. coli preparation and mesalazine compared to two studies [57, 58] in the maintenance of remission of ulcerative colitis. 120 patients with inactive ulcerative colitis were included in the first study. 11.3% of the patients treated with mesalazine relapsed after 12 wks as compare to that 16% who were treated with probiotics. The second study had 116 patients which also showed that the preparation of probiotic was effective as mesalazine in remission inducing and relapse prevention [58]. Many studies in Europe are testing the probiotics effect on inflammatory bowel disease.

2.5. Irritable Bowel Syndrome

This syndrome is described by a stomach torment, change in the habit of bowel and bloating, with a nonattendance of any clear mucosal abnormality and flatulence [59]. Probiotics also including Bifidus milk or acidophilus relieve constipation in some patients which were not controlled [60]. In a random placebo-controlled study which comprises 34 patients, Maupas, et al. [61] observed that S. boulardii reduced functional diarrhea but did not increase other symptoms of bowel syndrome. Halpern and their team [62] suggesting in random, crossover, double blind that heat killed lactobacilli administration for 6 weeks was more effective than placebo in irritable bowel syndrome symptoms relieving. They Hentschel, et al. [63] suggest the efficacy of preparation of 2 probiotics having E. coli and lactobacilli in 126 patients affected by non-ulcer dyspepsia and didn’t observe amelioration. S. boulardii minimize the duration of diarrhea activated by tube feeding in 3 trials [64-66]. Recently the study was double blind and compared the placebo in 128 critically ill patients of tube-fed with 2g of S. boulardii/day [66]. Probiotic treatment reduced the percentage of diarrhea days experienced by patients from 18.9% to 14.2% (p=0.007). The open 2 study results that lactobacilli may have efficacy against the overgrowth of small intestinal bacteria [67, 68] but S. boulardii is not effective in a random placebo controlled study [69]. Diarrhea is an adverse nearly constant effect of pelvic irradiation. A control randomized study showed a decrease in patients with diarrhea during pelvic irradiation by [70] in those patients receiving L. acidophilus NDCO 1748. Some past Open trails have been conducted in which freeze dried lactic acid bacterial cultures were used for the same symptoms [35] such therapeutic effects need to be further studied [71] discovered that S. boulardii may have effect when taken in high doses in some patients with chronic diarrhea related with HIV and before conclusions further evaluation should be drawn [71].

In the treatment of IBS, the meta-analysis of clinical features of probiotics was led by Moayyedi and his colleagues. In this study, 18 randomized clinically controlled trials including 1,650 IBS suspects were identified and the examining products include Streptococcus (1 study), Lactobacillus (6 studies), Bifidobacterium (3 studies), and different combination products (9 studies) (and one trail was reported on both Bifidobacterium and Lactobacillus). Out of these ten (n= 918) concluded results as a variable dichotomous, in these studies IBS symptoms were reduced by probiotics (persisting risk of symptoms in treatment group 95% CI (0.57-0.88) and with number treat of four needed. Total 15 trials reported results as a continuous variable, grouping for meta-analysis. These trials examined that probiotics had a highly significant effect on improvement of IBS symptoms as compared to placebo. Differences between different probiotics were shown in this meta-analysis with Bifidobacterium (2 trials involving 422 suspects), Streptococcus (1 trial involving 54 suspects), Lactobacillus (3 trials involving 140 suspects), and different combination products (four trials involving 302 suspects) in which all were showing benefits. In an improvement of individuals symptoms, probiotics had a good effect in improvement of bloating, flatulence, and pain sources [72].

In adults, varieties of studies showing that L. rhamnousus GG, B. infantis, and the different mixture of probiotics as L .rhamnosus LC705, L. rhamnous GG, Propionibacterium freudenreichii JS, and B. breve Bb99 are effective in minimizing the IBS symptoms. While in infants there is decrease in abdominal distention but there is no decrease in abdominal pain by L. rhamnosus GG [73-76].

2.6. Allergic Diseases

The reaction of hypersensitivity initiated by mechanisms of immunology is known as allergy, on the basis of immunological mechanisms that are involved the allergy is classified into non IgE-mediated allergy and IgE mediated allergy [77]. The allergic disease development is related to Microbiota aberrancies [78, 79]. In infants atopy development is detected due to reduced ratio of Bifidobacteria to Clostridia [78] and patients with allergy are often more colonized with Staphylococcus and Clostridium and have few Bifidobacterium and Enterococcus than in non-allergic persons [78, 80, 81]. Composition differences in gut micro-biota may manipulate the manifestation and development of atop. Higher risk of eczema development has been assigned to early colonization of Escherichia coli. Clostridium difficile is associated with recurrent wheezing, allergic sensitization in infants and eczema [82].

Disease of atopy arises from abnormal responses of the immune system to allergens present in the environment which leads to inflammatory allergic response [83]. An allergic skin disease known as Atopic dermatitis (AD) is widely common in kids from Western Europe and the US [84]. Such patients suffering from this disease have a maximum number of Clostridium and S. aureus in their colon and have a minimum number of Bacterioides, Enterococcus, and Bifidobacterium. Healthy intestinal microbiota with high recognition of importance with a value of the active role of probiotics in the treatment and prevention of human allergic disease in clinical trials.50% of reduction occurred in atopic eczema by administration of Lactobacillus GG to high risk infants [78]. In other experiments, children were treated for two months with a whey formula having L. rhamnosus or B. animalis subspecies lactis in Finland which resulted in improved skin condition. Similarly, results obtained L. rhamnosus plus L. reuteri were curative [85, 86]. In western society spread of atopic diseases has been increasing progressively. Hygiene hypothesis of allergy came up with the less exposure to microorganisms at an early age [87]. The most effective and earliest source of this type of exposure is related to the settlement of gut microflora. In human allergic disease, the probiotics regulatory role was first highlighted in the demonstration of the inhibition effect on lymphocyte’s proliferation and in vitro interleukin 4 generation [88, 89]. The immune-inflammatory response in allergic individuals to dietary antigens were minimized by probiotics, this partly increases the production of anti-inflammatory cytokines interleukin 10 [90] and also the transforming growth factor beta [91] which partly control allergic inflammation of gut [85].

2.7. Cancer

Cancer has a great importance in human well-being because of its increasing frequency in developed nations. The techniques for effectual treatment of cancer under study are incalculable and not long ago, the utilization of LAB has shown up in this field. Disease improvement and movement have been comprehensively related with unending irritation procedures created by outside components, for example, contamination, radiation, uneven eating regimen, heftiness, tobacco or the introduction to other ecological poisons [92].

The immune system and endogenous flora play role in carcinogenesis modulation. Probiotics may influence both of them which lead to investigating the probiotics role in curing and preventing animal tumors. Different authors have shown that some probiotics may minimize the enzymes, secondary bile salts and mutagens fecal concentrations which might be involved in carcinogenesis of colon [93]. Not all but some epidemiological studies suggest that fermented dairy products might have a protective effect against cancer or large colon adenomas [94</a>] LAB organisms are used for the prevention of colorectal cancer has been found mainly using colorectal cancer murine models [92]. One sample used Bifidobacterium adolescentis which expressed a recombinant endostatin demonstrated how this protected vector could specifically repress angiogenesis and tumor development in tumor mice models after its intravenous administration [95].

In a controlled double blind study with 138 patients in 1995, L. casei shirota preparation had preventive effect on the rate of bladder cancer after surgery [96]. In a variety of animals models involving rats and mice which were fed on oligofructose and/or insulin reduce the fecal water genotoxicity [97]. The numbers of precancerous lesions which are induced chemically are decreased [98, 99] and also defense function is stimulated. The activity of NK-cells and IL-10 was also increased [100]. Tumor incidence in organs (breast cancer in mice and rats, in lungs [101] and large intestine [102] were decreased by the addition of 5 to 15% oligofructose or inulin to the diet in the long term. This impact was significantly more proclaimed when a mix of prebiotics and probiotics were given [103]. In the colon of male Sprague-Dawley rats treated with 1,2-dimethylhydrazine, Xylooligosaccharide appeared to decrease the number of aberrant crypt foci [104].

2.8. Adverse Side Effects of LAB

LABs are (GMOs) genetically modified organisms and are well established in industries such as food industries and are widely accepted. For its use as a therapeutic agent, important regulatory concerns are needed to be addressed. LABs are specifically based on expression systems having selection markers as antibiotic resistance genes [105]. The transfer of antibiotics resistance of live vectors to intestinal microbiota has been discovered but this rare event has not reported in this field, however, this issue is important to be considered. LAB as live vectors opens an interesting and vast field of possibilities but measures related to regulation has to be considered to ensure the used strain safety. Some innovative selection markers and alternative up till now have already developed, some of them are tested successfully and positively evaluated by different health authorities [105-107]. These safe and alternative selection markers must be explored further that some of LAB adverse effect has been reported [108-110]. This shows that having positive therapeutic effects of such microorganisms and minimum adverse effect are registered they are not safe completely. This information should be given importance as the adverse effects observed were in high risk groups like immune compromised and critically ill patients, hospitalized patients and severely sick infants. The main harmful effects of LAB described are fungemia, GI ischemia and Sepsis [109]. In vulnerable populations LAB interfere with microflora forming opportunistic infections and leads to fungemia, bacteremia or different medical complication. Other strong evidences shows that using LAB as probiotics has anti-inflammatory effects however reports also show pro-inflammatory effects caused by group of bacteria which means that effect of LAB as probiotics is strain dependent, acting as a factor to be the therapeutic applications of host strains [111, 112].

2.9. Obesity

Obesity is seen as one of the real current general health issues and its effect is the most noteworthy in kids, adding to critical dreariness in adulthood [113]. The progress of metabolic complications connected with obesity amid youth track into adulthood and builds the danger for diabetes type 2 and early cardiovascular illnesses [114]. Dangerous elements of obesity in infant involve eating routine, low financial status, maternal obesity, quick outset weight pick up, and diminished physical activity. Additional to these dangers, a few reports have proposed that the gut microbiota is an imperative element influencing energy transfer and capacity in adipocytes [115-118]. Moreover, a late report demonstrated that newborn children with high quantities of the Bifidobacterium and low quantities of Staphylococcus may be shielded from overabundance weight gain amid later life [119]. The Bifidobacteria populace (and most different life forms in the family of Firmicutes) is somewhat lower in people with obesity than in slim individuals [120]. A comparative finding was accounted for in patients with type 2 diabetes mellitus in correlation with no diabetic patients [121]. These discoveries recommend that Bifidobacteria may have an impact on the advancement of obesity and its related independently existing medical condition. At the point when prebiotics like insulin-type fructans was given to mice, these were utilized as vitality substrates by microorganisms [122-124]. The quantity of Bifidobacteria expanded altogether, and there was a backward connection with the levels of lipopolysaccharide, glucose resistance and advancement of fat mass [124, 125]. Additionally, it has been accounted for how weight control plans in view of a high admission of protein and/or low admission of sugar or low fat utilization may change the composition of microbes and activity in the large intestine and consequently affect gut health. Modification of Micro-biota by utilization of probiotics may offer new headings for preventive and restorative applications in lessening the danger of obesity and overweight [115, 122, 125, 126].

2.10. Constipation

There are a significant number of reports on the constructive outcomes of fermented dairy items on constipation [127]. Unfortunately, greater parts of the studies were performed under inadequately controlled conditions. Positive results have been accounted for in studies with geriatric patients [128]. A double-blind crossover investigation of 50 patients with periodic constipation likewise indicated positive results. They got 1 g of a LAB preparation (containing 10 L. casei per gram) three times each day. Amid the treatment time of 30 days, either the medication or a fake treatment was regulated orally. The general effectiveness rate was 67% [129].

3. CONCLUSION

Probiotics are helpful and are progressively utilized for an assortment of Gastro-intestinal issue. Probiotics seem to adjust intestinal micro-flora and may apply their effect(s) by different mechanisms. Numerous types of probiotics exist and it is, by and large, acknowledged that all probiotics are not made equivalent. Efficacy may be because of a solitary strain or numerous strains or a mix of distinctive. Probiotics diminish the span of indications in intense infectious diarrhea. At the point when added to standard treatment, probiotics don't give extra benefit compared to standard treatment alone. Introduction of probiotics orally has appeared to fortify the different lines of the gut defense system. Probiotics likewise fortify non-particular host resistance to microbial pathogens and in this manner guide in their annihilation. The utilization of probiotics right now lies in lessening the danger of infections connected with gut barrier brokenness. Probiotics treatment has effectively advanced in the treatment of a number of conditions. Before bringing probiotics into routine use, legitimate assessment of these items is crucial.

Funding: This study received no specific financial support.

Competing Interests: The authors declare that they have no competing interests.

Contributors/Acknowledgement: All authors contributed equally to the conception and design of the study.

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