Everyone may contact toxic elements in various ways, such as in air, food, drinking water, daily necessities, and soil. The toxic elements not only cause environmental pollution but also cause great damage to human bodies. Several diseases are related to the amount of toxic elements accumulated in one’s body. For example, Cadmium (Cd), Beryllium (Be), and Arsenic (As), are reported as carcinogens.

Food supply quantity data 2013 released by Food Agriculture Organization of the United Nations (FAO) indicates that Taiwanese consume 35.41 kg seafood per capita per year. The average of sea food consumption in Taiwan is equivalent to double around the world. Thus, the more sea food a person consumes the more heavy metals a person accumulates, such as Cd and Hg. According to 2016 Environmental Performance Index (EPI) report launched at World Economic Forum (WEF), half of the world’s population are exposed to unsafe air quality. Toxic elements are omnipresent in our life. Therefore, how to reduce the concentration of the toxic elements in one’s body effectively becomes a very important issue.

Currently, the chelation therapy, including EDTA, DMSA, and DMPS, is the only way to excrete the toxic elements immediately from the body. However, it may have serious side effects, such as reducing the amounts of other essential minerals (Ca, Fe, and Zn, etc.) at the same time, dehydration, liver failure, renal failure, hypoglycaemia, and coagulation insufficiency. Hence, this treatment is not always suitable for patients. Recently, more and more researches have aimed at exploring other ways to eliminate toxic elements from the environments and animals. However, there are not such many studies to find the treatments in human bodies. Among the toxic elements, Cd, Hg, Pb, and Be are toxic heavy metals. Due to the different influence factors, such as different types of work, eating habits, and different location, the concentration of toxic elements in each individual varies.

The World Health Organization (WHO) has defined probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Probiotics are considered as a functional food. They can modulate the immune system functions, improvement of the barrier functions of the gut mucosa, and to suppress the growth of pathogenic bacteria [1]. The probiotics are highly used in clinical, including Diabetes, Hypertension, Urogenital health care, Lactose intolerance, Crohn’s disease and ulcerative colitis, Colon cancer, Inflammation, Immune function and infections, Peptic ulcer, Liver diseases, Food allergy, Upper respiratory tract infection, Oral health, and so on [2]. In recent years, some research studied the function of LABs (lactic acid bacteria) that removes heavy metals (toxic elements) in culture medium, animals, and human bodies.

LABs, such as Lactobacillus gasseri and Lactobacillus reuteri (L.reuteri), etc. can bind with the toxic metals (e.g. Cd) in vitro [3]. L. reuteri Cd70-13 and Pb71-1could remove Cd (25%) and Pb (59%) from the MRS culture medium; therefore, they may adsorb the toxic heavy metals in the intestinal tract [4]. In the water, exopolysaccharide (EPS) of Lactobacillus rhamnosus (L. rhamnosus ) can remove Al+3 and Cd+2 [5]. Bifidobacterium logum (B. Longum ) 46 is also able to remove Cd and Pb from the water [6].

In some animal studies, supplying Lactobacillus plantarum (L. plantarum ) CCFM639 can reduce the damage of liver and kidney [7]. It can also reduce the absorption of Al in the intestines and the accumulation of Al in the tissues, and discharged them from the faeces [7]. L. plantarum CCFM8610 can prevent heavy metals’ (Cd) acute poisoning [8,9]. L. plantarum and Bacilus coagulans (B. coagulans ) can treat and prevent Hg poisoning in high-risk areas in rats [10]. Pb can be absorbed 90% in the Lactobacillus acidipiscis (L. acidipiscis ) ITA44 and Lactobaciilus pentosus (L. pentosus ) ITA23 culture medium. Feeding L. acidipiscis ITA44 and L. pentosus ITA23 to broiler chickens can decrease Pb accumulation for 48% in liver and 28% in serum samples [11]. It is reported that pregnant women taking probiotic yogurt (L. rhamnosus GR-1) are able to control the levels of Hg and As in blood [12]. Among all the research, different probiotic species or strains have different effects on toxic elements in different media. Based on the literature, the purpose of this study is to verify whether a specific toxic element can be excreted from the human body by taking some specific probiotic strain for at least six months.

Features of this study include probiotic strains of participant supplement, gender, age, disease, and twice detecting value of concentration of toxic elements in hair. In this study, we want to answer that probiotics can remove or not, and what will be the important factor in the toxic element elimination from the human body.

Participants

The data in this study was providing by Bio Ray Biotech Inc. including 319 participants. Figure 1 shows the way how the data be collected. In this study, every participant must meet the following requirements: (1) The age of the participant must exceed 20 years old. (2) The participant has no intensively taken probiotics before. (3) The participant is required to take 20 billion CFU of a specific probiotic strain per day for at least six months. (4) The concentration of toxic elements (Cd, Hg, Pb, Be, As, Al) is duplicate measured before and after probiotics supplement. The numbers of participants in each probiotic strain are randomly assigned as follows: Lactobacillus paracasei BRAP-01 (AP01, Bio-Ray, Kaohsiung, Taiwan): nLP=114; Bifidobacterium longum BR022 (Bio-Ray, Kaohsiung, Taiwan): nBL=25; Lactobacillus acidophilus AD300 (Biena, Quebec, Canada): nLA=66; Lactobacillus reuteri BR101 (Bio-Ray, Kaohsiung, Taiwan): nLRe=69; Lactobacillus rhamnosus AD500 (Biena, Quebec, Canada): nLRh=45. The data of gender, ages, and disease states were collected following the ethical principles of Declaration of Helsinki and approved by the Research Ethics Committee for Human Subject Protection of National Chiao Tung University. The average of age and the distribution of genders and diseases of the study population were shown in Table 1. Diseases of participants were simply classified as four groups including cancers, diabetes (DM), hypertension (HTN) and he others.

Figure 1: The flow chart of data collection.

Table 1: Member of participants for each probiotic strain in different type.

Analysis of the toxic elements in hair

Each participant’s hair is used as the sample to detect the concentration of toxic elements, and the experiment is conducted by La Belle Vie Inc. First, 75 mg hair sample is collected, and then cleaned by organic solvents and interacting agents. The hair sample is later dissolve into a solution and analysed by using ICP-MS to detect the concentration of toxic elements. The unit used in this study is ng/g hair (ppb).

Statistical methods

Exact McNemar’s test, Wilcoxon signed-rank test, and regression tree are used in data analysis. In the following statistical analysis, significant level was set as 0.05. The statistical calculation was done in R version 3.2.4 (2016-03-10) and the related packages are “exact 2 × 2”, “MASS”, “exactRankTests”, and “rpart”.

Number of people with an abnormal concentration of mercury and beryllium was significantly reduce after probiotics supplement

First at all, we analysed the concentration distribution of presupplement and post-supplement value for each toxic element. It showed only the median of Hg is decreased after probiotics supplement (Table 2 and Figure 2).

Table 2: The concentration of toxic elements in hair for pro-post detection. ^apre: the concentration of toxic element before taking probiotic. ^bpost: the concentration of toxic element after taking probiotic. ^cThe unit of Median, MAD (median absolute deviation), Min (Minimum), and Max (Maximum) are ppb.

Figure 2: Boxplot for every toxic element pro-post supplement value. Red line: The normal ranges suggested by La Bella vie Inc.

To further investigate the changes of numbers from abnormal to normal ranges, we use the normal ranges suggested by La Belle Vie Inc. according to the investigation of hair elements evaluation in 5846 Japanese. Accordingly, the normal ranges of every element in hair were described as followed. (1) Cd is below 22 ppb. (2) Hg is below 6946 ppb. (3) Pb is below 1218 ppb. (4) As is below 89 ppb. (5) Be is below 0.74 ppb. (6) Al is below 7964 ppb. The number of concentration of toxic elements both in normal and abnormal cases were shown in Tables 3-8. The results demonstrate that the number of abnormal cases in Hg (p-value=0.001686) and Be (p-value=1.942 × 10^-5) decrease significantly after the participants have taken probiotics for at least six months. The intra-person difference showed that more than 50% participants who had decreased concentration of Hg, Pb, and As after taking probiotics for at least six months and 50% participants who is decreased the concentration of Be (Figure 3). Therefore, the intraperson decreasing effect of these toxic elements will be more attention.

Table 3: Contingency table of cadmium (Cd). aExact McNemar’s Test.

Table 4: Contingency table of mercury (Hg). aExact McNemar’s Test, **p-value<0.01.

Table 5: Contingency table of lead (Pb). aExact McNemar’s Test.

Table 6: Contingency table of arsenic (As). aExact McNemar’s Test.

Table 7: Contingency table of beryllium (Be). aExact McNemar’s Test, ***p-value<0.001.

Table 8: Contingency table of aluminum (Al). aExact McNemar’s Test.

Figure 3: Boxplot for the difference between pro-post detecting values for every toxic element. Red line: No difference of the concentration in the toxic element after taking probiotic for at least six months.

Different probiotics strains exhibited different abilities in toxic element elimination

By analysing the difference of toxic element levels before and after supplement of probiotics in each participant, it showed that the concentration of Hg (p-value=1.846 × 10^-5) and Be (p-value=0.0275) are decreased significantly after taken probiotic for at least six months (Table 9). Especially, people who have taken L. paracasei BRAP-01(pvalue= 0.01964), B. longum BR022 (p-value=0.02938), and L. reuteri BR101 (p-value=6.195 × 10^-6). In addition, concentration of Be decrease significantly after the participants have taken L. acidophilus AD300 (p-value=0.006438) for at least six months.

Table 9: Information about the difference of twice detection of the concentration of toxic elements in hair by taking each probiotic strain. ^aWilcoxon signed-rank test. *p-value<0.05.**p-value<0.01. ***p-value<0.001.

The concentration decreasing effects in specific group of regression tree

Furthermore, we use the regression tree to determine the rank of factors in the database. Every regression tree is designed as the terminal nodes which contains at least ten samples and the maximum depth of regression tree is three. The regression tree is based on the difference between pre-detecting and post-detecting toxic element concentration value which factor include gender, age, disease (cancer, DM, and HTN) and five probiotics strains. After regression tree generating, do Wilcoxon signed-rank test for every node in regression tree. Then, find which specific group have significant decrease of concentration and observe whether there is any interaction for every toxic element.

Figure 4A demonstrated the regression tree for the difference between pre-detecting and post-detecting concentration of Hg. The most important factor of significant decreased of the concentration of Hg is cancer (yes or no). The second important factor is probiotics strain. The third important factor is age (age more than or equal to 54 years old or age less than 54 years old). Three important factors are interacting with each other. In the regression tree of Figure 4A, Node3 shows that having taken probiotics for at least six months, participants not suffering from cancer were significantly decreased the concentration of Hg (p-value=1.352 × 10^-6); Node4 shows that participants not suffering from cancer and having taken probiotics which are L. paracasei BRAP-01, L. acidophilus AD300 or L. rhamnosus AD500 for at least six months significantly decreased of the concentration of Hg (p-value=0.01225); Node5 shows that participants not suffering from cancer and having taken probiotics which are B. longum BR022 or L. reuteri BR101 for at least six months significantly decreased of the concentration of Hg (p-value=1.431 × 10^-6); Node6 shows that participants not suffering from cancer, having taken probiotics which are B. longum BR022 or L. reuteri BR101 for at least six months and aging greater than or equal to 54 years old significantly decreased of the concentration of Hg (p-value=0.005602); Node7 shows that participants not suffering from cancer, having taken probiotics which are B. longum BR022 or L. reuteri BR101 for at least six months and aging less than 54 years old significantly decreased of the concentration of Hg (p-value=4.951 × 10^-6).

Figure 4: The regression tree for the differential concentration of mercury and beryllium in hair.

Figure 4B is the regression tree for the difference between predetecting and post-detecting concentration of Be. The most important factor of significant decrease in concentration of Be is gender (male of female). The second important factor is age. The third important factor also is age. Those important factors are interacting with each other. In the regression tree of Figure 4B, Node3 shows that males having taken probiotics for at least six months significantly decreased of the concentration of Be (p-value=0.0372); Node5 shows that males having taken probiotics for at least six months and aging greater than 50 years old significantly decreased of the concentration of Be (pvalue= 0.003893); Node7 shows that males having taken probiotics for at least six months and aging greater than 50 years old and less than 58 years old significantly decreased of the concentration of Be (pvalue= 0.005324). In this study, only Hg and Be concentration decreased significantly whereas other toxic elements do not show any specific effect of decreasing in this study.

The use of bacteria as bio sorbent in industrial and environment pollution has been studied for a long time. However, there are only few articles describing the use of probiotics as detoxification agent for heavy metal accumulation in animals. Present study is the first large scale investigation of the impact of probiotics on long term toxic accumulation. Also, it is the first time to compare the effect of different probiotics strain on the accumulation of different toxic element in human hair samples.

We found that the mercury accumulation is decreased in three of five different strains, including L. paracasei BRAP01, B. longum BR022 and L. reuteri BR101. Whereas the decision tree also showed that the supplement of B. longum BR022 and L. reuteri BR101 is also an important factor for the decreased mercury accumulation in the study population. The same result was also found in Bisanz’s study in Tanzania which means mercury may be a major target of probiotics as bio sorbent. However, the L. rhamnosus strain we used did not show the same effect of L. rhamnosus GR-1 which means the effects may be strain restricted. The same phenomenon was also observed by Kirillova [13] and Bhakta [4] that different strains of the same species may have different toxic element absorption abilities. Unlike lead and cadmium, the mechanism of mercury to bacteria interaction has not been well identified. The only one studied of mercury binding protein in lactic acid bacteria was demonstrated by Kinoshida [3], but the mechanism is still not fully understood. In the other hand, the bioremediation

In the other hand, the bioremediation of lead and cadmium was not observed in all the five strains in this investigation. It also agree with the results of Bisanz’s study in Tanzania that the same strain used in Bisanz’s study even showed a good lead and cadmium binding abilities in vitro [14]. It suggests that the protection effects of probiotics to decrease toxic element accumulation in human hair may be different from the simple in vitro removing tests. The mercury metabolic enzyme, like methylmercury demethylase may also play an important. All above, the right strain that prevent accumulation of toxic element in human should be confirmed by clinical trial or other in vitro experiments.

Another interesting finding in this study is demonstrating that cancer may be an important factor in mercury excretion. One of the hypotheses is that the use of traditional Chinese medicine is very common in Eastern Asia to treat cancer patients. The heavy metal contamination, especially mercury and arsenic, are found either in commercial or unknown source of traditional Chinese medicine very often [15,16]. Another possible cause is the high consumption of selenium in cancers [17-19], and selenium is an important factor in mercury detoxification [20-22].

Up to date, the significant decrease in the beryllium after probiotics uptake is the first time being discovered in present study. Unlike other toxic element, the researches of interaction between beryllium and probiotics are relatively rare. However, there is lacking of strong evidence between beryllium and lung cancer, the long term exposure to beryllium still causes the damages of the body, including berylliosis and skin and eye irritation [23-25]. Our data demonstrated that the beryllium decrease could be observed in female, 50-58 years old after uptake L. acidophilus AD300. Considered of the residence, genders and habits of study population, we hypothesis that smoking is the most possible route of beryllium accumulation since tobacco often contains trace amount of beryllium [26-28]. Altogether, this study implies that the supplement of certified probiotics strains could be used in treatment of chronic accumulation of toxic elements, especially the mercury and beryllium, in human bodies and may prevent further damages and diseases.