Lactobacillus plantarum CCFM639 alleviates

Appl Microbiol Biotechnol DOI 10.1007/s00253-015-7135-7

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Lactobacillus plantarum CCFM639 alleviates aluminium toxicity Leilei Yu 1 & Qixiao Zhai 1,3 & Xiaoming Liu 1 & Gang Wang 1 & Qiuxiang Zhang 1 & Jianxin Zhao 1 & Arjan Narbad 3,4 & Hao Zhang 1,3 & Fengwei Tian 1,3 & Wei Chen 1,2,3

Received: 22 August 2015 / Revised: 25 October 2015 / Accepted: 30 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Aluminium (Al) is the most abundant metal in the earth’s crust. Al exposure can cause a variety of adverse physiological effects in humans and animals. Our aim was to demonstrate that specific probiotic bacteria can play a special physiologically functional role in protection against Al toxicity in mice. Thirty strains of lactic acid bacteria (LAB) were tested for their aluminium-binding ability, aluminium tolerance, their antioxidative capacity, and their ability to survive the exposure to artificial gastrointestinal (GI) juices. Lactobacillus plantarum CCFM639 was selected for animal experiments because of its excellent performance in vitro. Forty mice were divided into four groups: control, Al only, Al plus CCFM639, and Al plus deferiprone (DFP). CCFM639 was administered at 109 CFU once daily for 10 days, followed Leilei Yu and Qixiao Zhai contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7135-7) contains supplementary material, which is available to authorized users. * Fengwei Tian [email protected] * Wei Chen [email protected] 1

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China

2

Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, People’s Republic of China

3

UK-China Joint Centre on Probiotic Bacteria, Norwich NR4 7UA, UK

4

Gut Health and Food Safety Programme, Institute of Food Research, Norwich NR4 7UA, UK

by a single oral dose of aluminium chloride hexahydrate at 5.14 mg aluminium (LD50) for each mouse. The results showed that CCFM639 treatment led to a significant reduction in the mortality rates with corresponding decrease in intestinal aluminium absorption and in accumulation of aluminium in the tissues and amelioration of hepatic histopathological damage. This probiotic treatment also resulted in alleviation of hepatic, renal, and cerebral oxidative stress. The treatment of L. plantarum CCFM639 has potential as a therapeutic dietary strategy against acute aluminium toxicity. Keywords Aluminium toxicity . Aluminium binding . Antioxidative activity . Lactobacillus plantarum

Introduction Metal pollution is an ongoing concern as it is the foundation of numerous health problems. Aluminium (Al) is the third most prevalent element in the earth’s crust, behind oxygen and silicon, and the most abundant metal after iron and calcium (Crisponi et al. 2012). It is found in drinking water due to its action as a flocculant is a common additive in various processed foods, is added to cosmetics of many types, and, increasingly, shows up pharmaceutical and cosmetic products (Shaw and Tomljenovic 2013; Ganrot 1986). Al is widely distributed in the environment and extensively used in daily life that results in its frequent exposure to humans. Dietary absorption is a common path to Al accumulation in the body. The European Food Safety Agency (EFSA) has declared 1 mg Al/kg of body weight to be the tolerable weekly intake (TWI) (Aguilar HA et al. 2008). Dietary surveys in people from different countries, including the UK, Germany, Finland, Japan, and China, estimate that the overall daily intake of Al from diet, including additives, ranges from 14 to 280 mg/week (Sixty-seventh

Appl Microbiol Biotechnol

meeting of the Joint FAO/WHO, Expert Committee on Food Additives JECFA 2007). These levels markedly exceed the tolerable weekly intake. Moreover, a poisoning incident was reported in Hunan Province, China, caused by pollution from the Al plant in 2014 (http://ejatlas.org/print/chuangyuanaluminium-plant-pollution-in-hunan-china). Another polluting incident occurred in a village in the UK where an estimated 20,000 people may have been exposed to high levels of Al for several weeks (http://www.nature.com/news/ 2006/060417/full/news060417-10.html). In short, a significant number of people in many countries, especially children, are at a high risk of Al exposure (Krewski et al. 2007). Al accumulates in most of the mammalian tissues, including liver, kidneys, spleen, heart, blood, bone, and the brain (Ward et al. 2001). This accumulation has a major detrimental impact on hepatocellular integrity and function (Kaneko et al. 2004). The kidneys are also highly vulnerable to the nephrotoxic effects of Al during the normal processes of excretion (Mahieu et al. 2003). Moreover, Al remains one of the most studied toxins affecting the nervous system. Some experts believe that Al crosses the blood–brain barrier, forming deposits in the brain and contributing to the formation of Alzheimer’s-like neurofibrillary tangles (Sharma et al. 2009). Al exposure disrupts lipid membrane fluidity, perturbing iron, magnesium, and calcium homeostasis and causes oxidative stress (Mailloux et al. 2011). Previous studies have found that oxidative stress was an important mechanism of Al toxicity. Some chelators (such as deferroxamine (DFO) and deferiprone (DFP)), phytochemicals with antioxidant properties (curcumin (Kumar et al. 2009), resveratrol (Zaky et al. 2013), and caffeic acid (Yang et al. 2008)) have been found to decrease level of Al in the tissues and Al-induced oxidative stress. However, DFO and DFP have unavoidable side effects, including agranulocytosis, anorexia, and a worsening of hepatic fibrosis (Kruck et al. 1990; Olivieri et al. 1998). Natural compounds have fewer side effects, and some of them can be administered as a dietary supplement, but their extraction is complicated and expensive (Zhai et al. 2013). Therefore, novel and safe strategies with reduced side effects for treatment of Al toxicity are desired. Food-grade microbes that can be delivered to the gastrointestinal tract and that are capable of sequestering toxins present a safe and cost-effective intervention (Bisanz et al. 2014). Furthermore, previous researches showed that toxic metal have a direct impact on the gut microbiota in human and animals, especially the numbers levels of lactobacillaceae were increased (Breton et al. 2013a; Breton et al. 2013b). Therefore, lactobacillaceae have potential abilities to be against toxic metal. Besides, some lactic acid bacteria (LAB), including Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus brevis, can bind and remove toxic metals, such as cadmium, lead, and copper in vitro and in vivo (Abo-Amer

et al. 2012; Halttunen et al. 2007; Tian et al. 2012; Zhai et al. 2015a; Zhai et al. 2013; Zhai et al. 2015c). Apart from their capacities to bind toxic metals, lactobacilli are also known to have positive influence on human health including their antioxidative properties, a characteristic that may be important for the alleviation of Al toxicity (Kullisaar et al. 2003). In this study, we selected a novel probiotic LAB strain with good Al-binding ability and antioxidative property and evaluated its protective effects in tissues of mice subjected to acute oral Al exposure. Changes in Al ion concentrations in different areas of the body were monitored, and the biomarkers of oxidative stress were assessed, and we proposed possible protective mechanisms against Al toxicity.

Materials and methods Chemicals and their sources Kits used to measure the levels of malondialdehyde (MDA; Njjcbio A003 MDA kit), glutathione (GSH; Njjcbio A006 GSH kit), GSH peroxidase (GPx; Njjcbio A005 GPx kit), superoxide dismutase (SOD; Njjcbio A001 SOD kit), catalase (CAT; Njjcbio A007 CAT kit), and acetylcholinesterase (AChE; Njjcbio A024 AChE kit) were purchased from the Jiancheng Bioengineering Institute (Nanjing, China). Aluminium chloride (AlCl3.6H2O) and other analytical laboratory chemicals and reagents were purchased from the Sinopharm Chemical Reagent Company (Shanghai, China). Bacterial strains and culture Thirty LAB strains were used in this study, including Lactobacillus casei CCFM5, CCFM9, CCFM30 and CCFM236; L. rhamnosus GG (ATCC53103), CCFM237, and CCFM311; Bifidobacterium bifidum CCFM7 and CCFM16; L. plantarum CCFM47, CCFM231, CCFM232, CCFM240, CCFM241, CCFM307, CCFM308, CCFM309, CCFM639 (CGMCC9664), CCFM8610 (CGMCC6077) and CCFM8661 (CGMCC5494); L. brevis CCFM12 and CCFM135; L. reuteri CCFM14 (CICC6226); L. gasseri CCFM15; L. johnsonii CCFM134; L. kefir CCFM136; L. bulgaricus CCFM4 and CCFM29; L. helveticus CCFM310 and Lactobacillus CCFM78 and were obtained from the Culture Collections of Food Microbiology (CCFM), Jiangnan University (Wuxi, China). CGMCC9664, CGMCC6077, and CGMCC5494 were stored in China General Microbiological Culture Collection Center (CGMCC). All strains from the CCFM were isolated from fermented dairy products, fermented pickles, or the feces of healthy infants. CICC6226 was purchased from the China General Microbiological Culture Collection Center (CICC). ATCC53103 was purchased from the American Type Culture Collection (ATCC) and used


as a reference for all of the experiments in this study. Lactobacilli and bifidobacteria were cultured in de Man, Rogosa, and Sharpe (MRS) broth (Hopebio Company, Qingdao, China) at 37 °C for 18 h. All strains were subcultured twice before the experiment.

Aluminium-binding assay The aluminium-binding ability of the thirty strains was analyzed as previously described with minor modifications (Abou-Shanab et al. 2007). Each strain was cultured, and then harvested by centrifugation (8000×g, 20 min, 4 °C). The bacterial pellets were washed twice with ultrapure water and then resuspended in ultrapure water containing 5 mg/L Al ion. The bacterial concentration was adjusted to 1 g/L (wet weight). The samples were then incubated for 2 h at 37 °C (pH 4.5). After a centrifugation (8000×g for 20 min, 4 °C), a sample was taken from the supernatant for the analysis of residual Al. The background Al concentration in each strain was assessed by preparing corresponding cell pellets in ultrapure water instead of Al solution. After centrifugation, the pellets were collected and digested in concentrated HNO3 with a microwave digestion system. Al concentration was measured by flame atomic absorption spectrophotometry (Spectr AA 220; Varian). The final Al-binding activities of the strains were calculated by after substracting the Al content of the bacterial cells.

Bacterial resistance to aluminium The Al resistance of each strain was determined by measurement of the minimum inhibitory concentration (MIC) (AlHabsi and Niranjan 2012). Al solution was diluted to final concentrations ranging from 64 to 2048 mg/L in MRS broth. The minimum concentration of Al that completely inhibited the bacteria growth was established as MIC.

Determination of antioxidative activity of LAB strains Preparation of samples Intact cells of the LAB were harvested after incubation for 18 h. The cells were washed with and resuspended in phosphate buffer (pH 7.2), and cell numbers were adjusted to 109 CFU/mL and place in ice. The cell-free extracts (CFE) were prepared by ultrasonic disruption (VCX500; Sonics and Materials Inc., USA). Sonication was performed in 3–8s pulses for 15 min in an ice bath. The cell debris was then removed by centrifugation (8000×g, 20 min, 4 °C), and supernatant was stored as CFE (Morales and Vazquez 2004).

2,2-diphenyl-1-picrylhydrazyl radicals scavenging activity The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging abilities of LAB strains and CFE were measured according to the method previously described (Chen et al. 2014). Scavenging ability was calculated using the equation (1): Scavenging effect ð%Þ ¼ ½1−A517ðsampleÞ=A517ðblankÞ 100:

Hydroxyl radical scavenging activity The hydroxyl radicals scavenging activities of the LAB strains and CFE were measured as previously described with some modifications (Zhang et al. 2011). Scavenging ability was calculated using the equation (2): Scavenging effect ð%Þ ¼

A536ðsampleÞ‐A536ðblankÞ A536ðcontrolÞ‐A536ðblankÞ 100:

Reducing activity The reducing abilities of the LAB strains and CFE were measured as previously described with some modifications (Morales and Vazquez 2004). Lipid peroxidation inhibition The thiobarbituric acid (TBA) method was used to measure the capacity of the LAB strains and CFE to inhibit linoleic acid peroxidation (Lin and Chang 2000). The inhibition capacity was calculated using the equation (3): Inhibition effect ð%Þ ¼ ½1−A532ðsampleÞ=A532ðblankÞ 100:

Tolerance of LAB strains to simulated GI tract conditions The resistance of the strains to simulated stomach and small intestine juice were tested as previously described (Huang and Adams 2004). The survival rate was calculated using the equation (4): Survival rate ð%Þ ¼

log cfu N1 100 log cfu N0

where N1 is the total viable count of each strain after treatment with simulated GI juices, and N0 is the total viable count of each strain before treatment (Guo et al. 2009).


expressed in microgram per gram of tissue wet weight, microgram per deciliter of blood, and milligram per gram of feces wet weight.

Animal experiments Animals and treatments Adult female BALB/c mice (6 weeks old) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and housed at the Laboratory Animals Center of Jiangnan University (JN No. 20140514-0825-17). Mice were kept in a temperature- and humidity-controlled room that was equipped to maintain a 12-h light/12-h dark cycle. Standard commercial mouse food and sterile water were given ad libitum. All mice were housed in stainless steel cages for 1 week before the experiments began. As shown in Table 1, the mice were randomly divided into four groups: control, Al only, Al plus CCFM639, and Al plus DFP. Skim milk served as a vehicle for the administration of CCFM639. On the basis of previous studies in mice, the oral LD50 values were 1990 mg/kg for AlCl6.6H2O and 222 mg/kg for Al ion (Llobet et al. 1987). Therefore, a similar dose was selected for the present study. Before Al exposure, the body weight of all animals was measured at 23±2 g. Each animal was then dosed with 5.14 mg Al ion. At 0, 8, 12, 16, and 24 h after Al exposure, each surviving mouse was placed in a clean empty cage for 20 min, and the fecal samples were collected. All mice were then sacrificed under light ether anesthesia. Blood samples were collected, and the liver, kidneys, and brain were excised, cleaned, and stored at −80 °C. Determination of aluminium in tissues, blood and feces Samples were digested in concentrated HNO3 using a microwave digestion system (Zumkley et al. 1979). The Al concentration in the liver, kidneys, brain, blood, and feces was determined with a graphite furnace atomic absorption spectrophotometer (Spectr AAS or AA; Varian). Al content was Table 1

Animal experimental protocol

Group (n=10)

Control Al only Al+CCFM639 Al+DFP

Determination of MDA, GSH, SOD, CAT and AChE The levels of MDA and GSH and the activities of SOD, CAT, GPx, and AChE were measured using specific assay kits described in the BChemicals and their sources^ section and used according to the recommendations of the manufacturer. Histopathological studies The livers, kidneys, and brains were fixed in 10 % formalin saline for 48 h. The specimens were then embedded in paraffin and sliced into 5-μm thicknesses and stained with hematoxylin-eosin for examination by light microscopy. The results of the histopathological studies were analyzed with a semistatistical evaluation. Statistical analysis Results are reported as the mean±the standard error of the mean (SEM). A minimum of three independent experiments was carried out for each assay. The statistical significance of data comparisons was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Values for P