Safety Assessment of Bacillus licheniformis Me1 Isolated from Milk for Probiotic Application

Assay kits for determination of urea, creatinine, bilirubin, aspartate aminotransferase (SGOT), alanine amino transferase (SGPT), lactate dehydrogenase (LDH), creatinine kinase (CK Nac), and alkaline phosphatase (ALP) were obtained from Aspen Laboratories Pvt Ltd, India. Assay kit for analysis of glucose, cholesterol, and triglycerides (TG) were from Span Diagnostics Ltd, India. Kits for determination of sodium, potassium, and chloride were obtained from Coreal Clinical Systems, India. All other chemicals and solvents used for this study were of the highest purity obtained from Merck Pvt Ltd, India.

The culture B. licheniformis Me1 isolated from milk was used as the test article in this study. This culture was initially screened based on its potent antibacterial activity and subsequently identified using several biochemical and molecular techniques. (Nithya and Halami, unpublished data). The test article was cultivated in Luria Bertani (LB) broth for 36 hours at 37°C under shaking (150 rpm) condition. Subsequently, cells were harvested by centrifugation. The test article was prepared at a concentration of 1.1 × 10¹¹ colony-forming unit (CFU)/g of cell pellet for experimental feed preparation. The diet was prepared according to Reeves et al.²⁸ For acute and subchronic toxicity studies, AIN93M and AIN93G diets were used, respectively. For the subchronic study, diet was prepared once every 2 days with the test article. According to the body weight (BW) changes in the rat and feed intake changes, adjustments for the addition of test article in the diet were made. The experimental diet was stored at 4°C to ensure viability.

Animal care and handling conformed to the guidelines of the Committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India, and the protocols were approved by the Institutional Animal Ethical Committee (IAEC). The albino Wistar rats bred in the Central Food Technological Research Institute (CFTRI) animal house facility were housed in stainless steel cages (1 rat/cage) with a 12-hour light/dark cycle in a controlled atmosphere viz temperature 22°C ± 3°C and a relative humidity of 60% to 70%. The animals were fed ad libitum and had free access to drinking water during the experimental period. The drinking water was periodically analyzed and was determined to be free of contaminants. The animals were acclimatized to above experimental conditions for 5 days prior to the start of dosing for any toxicity study.

An acute toxicity study was performed in accordance with Organization for Economic Cooperation and Development (OECD) Guideline for the Testing of Chemicals No. 423; Acute Oral Toxicity—Acute Toxic Class Method, adopted December 17, 2001. Twelve healthy adult male albino Wistar rats (220 ± 10 g) were randomly assigned into 2 groups (6 per group), namely the control group which received the vehicle alone and the probiotic-treated group which received test article of 1000 mg/kg BW of rat, as a single dose. This dose level corresponds to 1.1 × 10¹¹ CFU/kg BW of rat and was selected based on previous references that have evaluated the in vivo safety of probiotic bacteria.^25,26,29 No female rats were used for acute toxicity study. Following the initial dose, the animals were observed with respect to general behavior, signs of toxicity, and mortality for 4 continuous hours and then twice per day over a period of 14 consecutive days. The general behavior, signs of toxicity include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, digestive, autonomic, and central nervous systems. Observations were also made on behavior patterns, such as tremors, convulsions, salivation, stool consistency, lethargy, sleep and changes in gait, posture, and response to handling. Daily feed and water intake was recorded throughout the experimental period and the BW of the rats were documented on days 0, 4, 7, 10, and 14. On the 15th day of the experiment, following an overnight fast, the animals were weighed and sacrificed with ether anesthesia. The blood samples were collected in sterile conditions by cardiac puncture in ethylenediaminetetraacetic acid (EDTA-2K) containing tubes for hematological analysis and in nonanticoagulant tubes for serum biochemical investigations. The vital organs were weighed and relative organ weights (g/100 g BW) were calculated. A gross pathological organ examination was performed. Histopatholological analysis (if necessary) was done for any treatment-related abnormalities.

Male and female albino Wistar rats bred in the CFTRI animal house facility were selected and randomly split into 3 groups, each comprising 6 males and 6 females, weighing 36 ± 1 g. The first group was kept as control and fed only the basal diet. Experimental diet consisting of basal diet supplemented with test article at 2 dose levels 100 and 1000 mg/kg BW/d (which corresponds to 1.1 × 10¹⁰ and 1.1 × 10¹¹ CFU) was fed to groups 2 and 3, respectively, for 13 weeks. Test diet and uncontaminated water were available ad libitum throughout the experimental period. At the end of the experiment, all surviving animals were fasted overnight before anesthetization with diethyl ether and sacrificed. The results were analyzed according to the OECD Guideline for the Testing of Chemicals No. 408; Repeated Dose 90-Day Oral Toxicity Study in Rodents, adopted September 21, 1998.

Adult male and female mice (Swiss Albino, Mus musculas, CFT strain) weighing 36 ± 2 g were obtained from our animal house facility. The study was conducted according to OECD Guideline for the Testing of Chemicals No. 474; Mammalian micronucleus test, Adopted July 21, 1997. Mice of each gender were randomly assigned into 4 groups and housed (5 animals in a single cage). The environmental condition in the experimental room was maintained viz temperature 22°C ± 3°C, relative humidity 60% to 70%, at a 12-hour light/dark cycle. The animals had free access to the standard diet³¹ and water, which were available ad libitum. Animals were acclimatized to the experimental conditions for 5 days before the start of the study. The animals were weighed and observed for signs of illness or other abnormalities at the start of the study. Ethane methane sulfonate (EMS; 5 μg/mL) and water were used as positive and negative controls, respectively. The test article, B licheniformis Me1 was administrated to 2 different mice groups at concentrations, namely, 1.1 × 10¹⁰ CFU/kg BW and 1.1 × 10¹¹ CFU/kg BW of mice, using water as a vehicle. The test article was given by oral route for 2 consecutive days at 24-hour interval. The positive and negative controls were given to the control groups mice only once by intraperitoneal injection on the last test article administration day. Thirty-six hours after the last day of the test article administration, blood samples from the mice were collected by tail trimming. Peripheral blood samples were smeared on acridine orange coded slides and observed under a fluorescent microscope. A minimum of 2000 reticulocytes were scored for the presence of micronuclei. The proportion of immature erythrocytes among total erythrocytes was determined for each test group and compared with control.

Male albino rabbits weighing 2500 ± 250 g from our animal house facility were used for the study. Animals were housed individually in metal cages at 20°C ± 3°C, relative humidity of 40% to 70% and at a 12-h light/dark cycle. Animals were fed ad libitum with conventional laboratory diet. Tap water was routinely analyzed for contaminants and was also available ad libitum. Three healthy male albino rabbits were used for acute eye irritation study. Both eyes of the animals were examined 24 hours prior to the start of the study. Only animals not showing any ocular defects or preexisting corneal injury were used in the experiment. The dosage of test article (0.1 g of the undiluted B. licheniformis Me1 cell mass at a concentration of 1.1 × 10¹¹ CFU/g) was placed into the conjunctival sac of the left eye of each rabbit. The untreated right eye served as the control. Eyes were not washed after the application. Eyes were examined for any lesions at 1, 24, 48, and 72 hours after the test article application. Any clinical signs of toxicity or signs of ill health of the animals were recorded during the study. At the end of the experiment, the weight of animals was determined. The study was performed in accordance with the OECD Guidelines for Testing of Chemicals No. 405; Acute Eye Irritation/Corrosion, adopted April 24, 2002. Eye irritation scores were evaluated according to the Draize (1977) and the OECD 405 (April 24, 2002) scoring systems.

The conditions for housing, feeding, and drinking of the rabbits were the same as described above. For the acute skin irritation experiment, 3 male albino rabbits were used. The fur was removed from a small area of the animal’s trunk, approximately 24 hours before the test. An undiluted dose of 0.5 g of the test article (corresponding to a concentration of 1.1 × 10¹³ CFU/g) was moistened sufficiently with water to ensure good contact with the skin and applied to a small skin area (approximately 6 cm²) of 3 animals and covered with sterile gauze patches, held in place with nonirritating tape. Untreated skin areas of each animal served as the control. After 4 hours of exposure, the test article was removed from the animal’s skin by washing with water. Animals were examined for erythema and edema at 1, 24, 48, and 72 hours after the removal of the test article. The test article was evaluated according to the Draize (1959) method (OECD 404, 2002) for any skin irritant effect. The study was performed in accordance with the OECD Guidelines for Testing of Chemicals No. 404; Acute dermal irritation/Corrosion, adopted April 24, 2002.

Statistical analysis of the data was performed with SPSS Software (version 16.0). Comparison of results between control and treatment groups of male and female groups were separately carried out by 1-way analysis of variance (ANOVA), and a post hoc analysis of individual pair difference was performed by Duncan multiple range tests. All the data are presented as mean ± standard error of the mean (SEM). A P value of < .05 was taken as statistically significant.

A 14-day oral acute toxicity study in adult male albino Wistar rats was performed to investigate the short-term effect of the test article, B. licheniformis Me1 administration. The results of this study provide useful preliminary toxicity data to determine appropriate dose levels for repeated-dose toxicity studies as well as for determining possible target organs to be examined, more closely in toxicity studies of a longer duration. No clinically related signs or any mortality were observed during the 14 consecutive days in the treated male rats. A single dose of 1.1 × 10¹¹ CFU/kg BW produced no treatment-related signs in behavior or changes in locomotor activity, respiratory, digestive, and circulatory or central nervous system, and autonomic activity. No abnormality was observed in skin, fur, and eyes and also no obvious signs of toxicity or any changes in other physiological activities immediately after administration or during the posttreatment period in any of the animals. Feed and water uptake were normal and there was no loss or gain in BW when compared to the control group. At necropsy, neither significant difference in the relative organ’s weight nor gross pathological alternations in the internal organs were found in all the treated and the control rats and hence, according to the OECD Guideline No. 423, histopathological examinations of organs were not carried out.

The micronucleus test was conducted to investigate the formation of micronuclei containing chromosome fragments or whole chromosomes and is considered the most reliable assay for cytogenetic damage. In the micronucleus assay, neither any differences in BW between the treatment groups compared to the control group nor any signs of toxicity were noted in clinical observations following administration of the test article at doses of 1.1 × 10¹¹ and 1.1 × 10¹⁰ CFU/kg BW. The ratio of reticulocytes to total erythrocytes was used an indicator for the evaluation of bone marrow toxicity. The results clearly demonstrate that the number of immature erythrocytes in each dose did not significantly increase above the concurrent negative (water) control frequencies. Additionally, it was always within the historical negative control range. As expected, animals in the EMS-treated positive control group showed a significant increase in the frequency of micronuclei compared to the negative controls. In contrast, no statistically significant induction of micronuclei in the test article fed groups was observed. The ratio of reticulocytes to total erythrocytes in the treated groups did not show any significant decrease as compared to the negative control group. For female mice, the average reticulocyte to total erythrocyte ratio in the negative control group was 2.17%. Also, the treated groups 1.1 × 10¹¹ and 1.1 × 10¹⁰ CFU/kg BW per day showed 2.07% and 1.93% of reticulocyte to total erythrocyte ratio, respectively, a reduction of 25.9% from the positive control group. In male mice, the negative control group showed a ratio of 1.85% and the 2 dose groups 1.1 × 10¹¹ and 1.1 × 10¹⁰ CFU/kg BW/d exhibited 1.51% and 1.10% reticulocytes to erythrocytes ratio. In the male group, the positive control showed 27.12% decrease in the ratio. These results clearly show that Me1 did not cause any signs of bone marrow cytotoxicity of mice in the range of the doses tested.

In the negative control groups, the incidence of micronucleated reticulocytes in the peripheral blood per 1000 reticulocytes was 1.18 ± 0.5 in males and 0.98 ± 1.1 in females. These results were within the historical reference range.²⁷ The positive control group had a statistically increased mean frequency of 26.14 ± 3 in males and 23.2 ± 5 in females as compared to the negative control group. The micronucleated reticulocytes per 1000 reticulocytes were found to be 1.03 ± 0.7 and 0.89 ± 1.2 in males and 1.2 ± 1 and 1.03 ± 0.9 in females at the test article dose levels of 1.1 × 10¹¹ and 1.1 × 10¹⁰ CFU/kg BW per day, respectively. Since the mice peripheral blood micronucleus assay did not show any statistically significant changes according to the OECD Guideline for the Testing of Chemicals No. 474, there is no indication that B. licheniformis Me1 administration caused any genocytotoxicity.

B. licheniformis Me1 cell mass application to the conjunctival mucosa of the eyes did not result in any test article–related irritant effect at any time during the experimental period. Furthermore, we did not observe any negative symptoms in either the cornea or the iris. According to the European Commission (EC) criteria of 2001/59/EEC for the classification and labeling requirements for dangerous substances and preparations, the test article is not required to be classified or labeled as an irritant to the eye.

B. licheniformis Me1 cell mass application to the skin did not reveal any clinical signs of erythema and edema at 1 hour after removal of the patch and until 72 hours later. According to the EC directive 2001/59/EEC, it is not required to classify or label the test article as a skin irritant.