In vivo safety assessment of bacteriocinogenic Bacillus licheniformis Me1shodhganga.inflibnet.ac.in/bitstream/10603/17837/9/14... · 2018-07-09 · Bacillus licheniformis Me1 performed

Chapter – 4

In vivo safety assessment of bacteriocinogenic

Bacillus licheniformis Me1

Chapter – 4

Safety assessment of Bacillus licheniformis Me1

160 Nithya V.

4.1. Abstract

In this Chapter, the in vivo toxicological safety assessment of the culture,

Bacillus licheniformis Me1 performed for food industry application has been

discussed. The in vivo tests performed included: acute and subchronic oral toxicity in

rat, micronucleus assay for detecting cytogenotoxicity in mice, and acute eye and skin

irritation studies in rabbits. The acute toxicity study performed in male albino Wistar

rats demonstrated no treatment-related illness or mortality. A 90-day subchronic oral

toxicity study was performed using two doses (1.1×1011

and 1.1×1010

CFU/kg BW).

In this study, data analysis of the body weight gain, food and water consumptions,

clinical observations, haematology, serum biochemistry, organ weight ratio and

histopathological findings did not show significant differences between control and

treated groups. These results proved the no-observed-adverse-effect level (NOAEL)

of B. licheniformis Me1 is greater than 1.1×1011

CFU/kg BW of rat. Acute dermal

irritation and acute eye irritation test conducted in rabbits showed no oedema or

erythema and ocular lesions, respectively. The micronucleus assay conducted in mice

revealed no signs of cytogenotoxicity. Since no toxicity was observed in any of the

toxicological studies, the culture B. licheniformis Me1 could be considered as safe for

use in food industry.

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4.2. Introduction

For decades, Bacillus and their metabolites have found several

biotechnological applications, including enzymes, amino acids, antibiotic production,

preparation of fermented foods and as pest control agents, etc. In addition, several

strains of Bacillus have also been shown to possess probiotic potentials (Hong et al.

2008; Kumprecht and Zobac 1996; Sanders et al. 2003). The use of Bacillus species

showing properties such as immune stimilation, antimicrobial activities and

competitive exclusion of pathogens, as a probiotic feed suppliment is increasing

rapidly (Caruso et al. 1993; Urdaci and Pinchuk 2004). For use as probiotics, there are

several imminent advantages of spore forming Bacillus spp. over LAB. The

underlying rationale for this perception is that Bacillus can survive in foods requiring

harsh processing conditions, such as high temperature and pressure, they survive

better under gastrointestinal tract (GIT) conditions, possess a long shelf-life and

remains viable throughout the shelf-life at room temperature and refrigerated

conditions (Cutting 2011). Furthermore, due to its better survivability, the effective

dose required for Bacillus as probiotic supplement is less in comparison with that of

LAB (Durkee 2012).

Many studies have documented the presence, diversity, stability and functional

properties of Bacillus spp. in fermented foods (Hyung et al. 2001; Inatsu et al. 2002;

Jeyaram et al. 2008; Sarkar et al. 2002). The use of antibacterial peptide- producing

Bacillus strains as starter culture provides an additional advantage for inhibiting

spoilage microorganisms (Jeyaram et al. 2008). Bizani et al. (2008) reported that the

antimicrobial compound cerein 8A produced by B. cereus 8A can be used as a

biopreservative in dairy products. Similarly, Bacillocin p490, a novel bacteriocin

produced by a thermophilic strain of Bacillus licheniformis was observed to control B.

smithii grown in water buffalo milk (Martirani et al. 2002).

Thus, a number of reports suggest that Bacillus strains including B. subtilis, B.

licheniformis, B. coagulans, B. clause and B. cereus can be consumed as probiotics

(Endres et al. 2009; Hong et al. 2008; Sanders et al. 2003; Sorokulova et al. 2008;

Urdaci and Pinchuk 2004). However, the use of Bacillus spp. for human consumption

raises question of safety. A number of species, such as B. anthracis, B. cereus, B.

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162 Nithya V.

thuringiensis, B. pseudomycoides and B. weihenstephanesis are known to be

pathogenic due to their ability to produce toxins. B. cereus produces enterotoxins and

emetic toxins and has been recognized as the major cause of food poisoning and other

non-gastrointestinal infections (Logan 2004). Spores of B. cereus could germinate and

produce enterotoxin in stored food (Nout et al. 1998). Furthermore, a number of B.

cereus probiotics have been shown to carry the enterotoxin genes (Hoa et al. 2000)

and one product, Paciflor, used in animal feed has been withdrawn from use in

European Union (SCAN 2001). However, not all B. cereus are pathogenic and their

pathogenicity varies with strains (Arnesen et al. 2008). In addition, B. thuriengiensis

commonly used as a bio-pesticide are also known to produce enterotoxins and cause

gastroenteritis (Jensen et al. 2002). The B. subtilis group (incl. B. subtilis, B.

licheniformis, B. pumilus and B. amyloliquefaciens) are usually considered relatively

safe, but are also reported to be involved in food-borne illness (From et al. 2005;

2007; Rowan et al. 2003; Salkinoja 1999). B. licheniformis occasionally is associated

with bovine toxemia and abortions (Johnson et al. 1994). However, the involvement

of species of B. subtilis group has been considered of little significance in food-

poisoning incidents. Another concern about the application of Bacillus in food

products is that, certain strains of Bacillus can cause opportunistic infections. Oggioni

et al. (1998) reported B. subtilis used for probiotic preparations caused septicemia in

immunocompromised patients. Therefore, the use of these spore forming bacteria as

dietary supplements, functional foods and for incorporation in pharmaceutical

products requires careful safety assessment using suitable models.

New species and strains of probiotic bacteria are becoming commercially

available as dietary ingredients and as functional foods and also being incorporated in

pharmaceutical products. Hence, it is very important that novel microorganisms,

which can be introduced into the human food chain, should be properly tested for its

safety and efficacy. Only limited information about the in vivo as well as in vitro

safety evaluation of Bacillus spp., viz, B. subtilis, B. coagulans, B. indicus, B.

licheniformis is available and these cultures are described to be safe for use without

having any adverse effects (Duc et al. 2004; Endres et al. 2009; Sorokulova et al.

2008). In previous section, (Chapter 3), the result of ABP potency of B. licheniformis

Me1 against wide range of food-borne pathogens was documented. In addition, the

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163 Nithya V.

ABP of this isolate was found to be stable at wide range of temperature (40 to 100°)

and pH (2 to 10) making it a most notable candidate for application as

biopreservatives. Thus, concerning the ability of the culture to produce potential ABP

and its safety and potential applications in food industry for biopreservation or as a

probiotic, an in vivo toxicological safety assessment of the bacterial culture B.

licheniformis Me1 was carried out using suitable animal models.

4.3. Materials and methods

4.3.1. Chemicals and reagent kits

Assay kits for determination of urea, creatinine, bilirubin, aspartate

aminotransferase (SGOT), alanine amino transferase (SGPT), lactate dehydrogenase

(LDH), creatinine kinase (CK Nac), alkaline phosphatase (ALP) were obtained from

Aspen Laboratories Pvt. Ltd., India. The test kit for determination of Na+/K

+ and Cl

- was

procured from Coral clinical systems, India. Assay kit for analysis of glucose, cholesterol,

and triglycerides (TG) were purchased from Span Diagnostics Ltd., India. Cellulose,

sucrose, cystine, choline bitartrate, butylhydroquinone and vitamin mix were obtained

from Himedia, India. The mineral mix was obtained from SRL, India. All other chemicals

and solvents used for this study were obtained from Merck India, Pvt. Ltd.

4.3.2. Preparation of test culture

The test culture, B. licheniformis Me1 was cultivated in LB broth for 36 h at

37°C under shaking (150 cycles/min) condition. Subsequently, cells were harvested

by centrifugation at 10000 g at 4°C. The test culture was prepared at a concentration

of 1.1×1011

CFU/g of cell pellet for experimental feed preparation.

4.3.3. Oral acute toxicity study in rats

4.3.3.1. Preparation of diet and dosage

For acute toxicity study, AIN93M diet was used for feeding rats and was

prepared according to Reeves et al. (1993). A detailed composition of the AIN93M

diet is given in the Table 4.1. The animal diet consisting of the test culture at a

concentration of 1000 mg/kg BW of rat was used for single dose study.

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Table 4.1. Composition of AIN93M diet

Ingredients % Mineral Mix: Calcium (0.5%), Phosphorus

(0.31%), Potassium (0.36%), Magnesium

(0.05%), Sodium (0.13%), Chlorine (0.20%),

Iron (39%), Flourine (1.0 ppm), Zinc (35 ppm),

Manganese (11 ppm), Copper (6 ppm), Cobalt (0

ppm), Iodine (0.21 ppm), Chromium (1 ppm),

Molybdeum (0.14 ppm), Selenium (0.22 ppm).

Vitamin Mix: Vitamin A (4 IU/g), Vitamin D-3

(1 IU/g), Vitamin E (78.8 IU/kg), Vitamin K (6

ppm), Thiamin hydrochloride (6 ppm),

Riboflavin, (6.5 ppm), Niacin (6.5 ppm),

Pantothenic acid (16 ppm), Folic acid (2.1 ppm),

Pyridoxine (5.8 ppm), Biotin (0.2 ppm), Vitamin

B-12 (28 mcg/kg), choline chloride (1,250 ppm),

Ascorbic acid (0 ppm).

Corn Starch 46.5692

Dextrin 15.5

Casein- Vitamin free 14

Sucrose 10

Powdered cellulose 5

Soybean oil 4

AIN93M Mineral Mix 3.5

AIN 93 Vitamin Mix 1

Choline Bitartrate 0.25

L- Cystine 0.18

t- Butylhydroquinone 0.0008

4.3.3.2. Animals and husbandry

Animal care and handling conformed to the guidelines of the Committee for

the purpose of control and supervision of the 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 CFTRI animal house

facility were housed in stainless steel cages (one rat/cage) with a 12 h light/dark cycle

in a controlled atmosphere viz. temperature 22 ± 3ºC and a relative humidity of 60-

70%. The animals were fed ad libitum and had free access to uncontaminated water

during the experimental period. The healthy albino Wistar rats were acclimatized to

experimental conditions for 5 days prior to the start of dosing for the toxicity study.

4.3.3.3. Acute toxicity study

An acute oral 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. This study was conducted in order to determine the dose-toxicity

relationship and to provide a dose standard for the sub acute oral toxicity study.

Detailed clinical observations were made prior to the exposure of the animals to test

culture. Twelve healthy adult male albino Wistar rats with a body weight ranging from

220 ± 10 g were used in this study. No female rats were used for acute toxicity study.

All the rats were randomly assigned into two groups (n = 6 per group), namely the

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control group which received just the basal diet and the treated group which received

the test culture at a concentration of 1.1×1011

CFU/kg BW of rat as a single dose. This

dose level was selected based on earlier literature reports that have evaluated the in vivo

safety of probiotic bacteria (Duc et al. 2004; Endres et al. 2009; Sorokulova et al. 2008).

Following the initial dose, the animals were observed with respect to general

behaviour, signs of toxicity and mortality for four continuous hours and then twice per

day over a period of 14 consecutive days. All the data were recorded systematically.

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 behaviour 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 was recorded on days 0, 4, 7, 10 and 14.

Following an overnight fast, on the day 15, all the tested animals were weighed and

sacrificed with ether anaesthesia. Blood was withdrawn (by cardiac puncture from

anaesthetized animals) under sterile conditions by cardiac puncture in ethylenediamine-

tetracetic acid-2K (EDTA-2K) containing tubes for haematological and in non-

anticoagulant tubes for serum biochemical investigations. The animals were then killed

humanely and the vital organs including liver, spleens, kidneys and heart were removed

under sterile conditions and processed for routine gross and microscopic examination.

The vital organs were weighed and relative organ weights (g/100g BW) were

calculated. A gross pathological organ examination was performed. Histopatholological

analyses (if necessary) were carried out for any treatment-related abnormalities.

4.3.4. Subchronic 13-week oral toxicity study in rats

4.3.4.1. Preparation of diet and dosage

For subchronic toxicity study, AIN93G diet was used and a detailed

composition of the AIN93G diet in given in Table 4.2. The diet was prepared

according to Reeves et al. (1993). The experimental diet consisting of basal diet

supplemented with the test culture at two dose levels 100 and1000 mg/kg BW of

rat/day (which corresponds to 1.1×1010

and 1.1×1011

CFU/Kg BW of rat) were

prepared once every two days. This dose levels were selected based on previous

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166 Nithya V.

references that have evaluated the in vivo safety of probiotic bacteria (Endres et al.

2009; Duc et al. 2004; Sorokulova 2008). According to the BW changes of the rat and

feed intake changes, adjustments for the addition of test culture in the diet were made.

The experimental diets were stored at 4ºC to ensure viability.

Table 4.2. Composition of AIN93G diet

Ingredients % Mineral Mix: Calcium (0.51%), Phosphorus

(0.32%), Potassium (0.36%), Magnesium

(0.05%), Sodium (0.13%), Chlorine (0.22%),

Iron (39%), Flourine, (1.0 ppm), Zinc (35 ppm),

Manganese (11 ppm), Copper (6 ppm), Cobalt (0

ppm), Iodine, (0.21 ppm), Chromium (1 ppm),

Molybdeum (0.14 ppm), Selenium (0.24 ppm).

Vitamin Mix: Vitamin A (4 IU/g), Vitamin D-3

(1 IU/g), Vitamin E (81.6 IU/kg), Vitamin K

(0.29 ppm), Thiamin hydrochloride (6.1 ppm),

Riboflavin (6.7 ppm), Niacin (30 ppm),

Pantothenic acid (16 ppm), Folic acid (2.1 ppm),

Pyridoxine (5.8 ppm), Biotin (0.2 ppm), Vitamin

B-12 (29 mcg/kg), choline chloride (1,250 ppm),

Ascorbic acid (0 ppm).

Corn Starch 39.7486

Maltodextrin 13.2

Casein- Vitamin free 20

Sucrose 10

Powdered cellulose 5

Soybean oil 7

AIN93G Mineral Mix 3.5

AIN 93 Vitamin Mix 1

Choline Bitartrate 0.25

L- Cystine 0.3

t- Butylhydroquinone 0.0014

4.3.4.2. Subchronic toxicity study

Male and female albino Wistar rats bred in the CFTRI animal house facility

were selected and randomly grouped (3 groups), each comprising of six males and six

females weighing 36 ± 1 g. Animals were maintained as described above (section

4.3.3.2). The first group was treated as the control group and fed only with the basal

diet. Experimental diet consisting of the basal diet supplemented with the test culture

at a concentration of 1.1×1011

and 1.1×1010

CFU/kg BW of rat was fed to the groups 2

and 3, respectively. Animals were dosed using this regime daily for 13 weeks. Test

diet and uncontaminated water were available ad libitum throughout the experimental

period. During the period of administration, the animals were observed twice daily

and weighed weekly to detect signs of toxicity. Daily visual observations were made

and recorded systematically, similar to those performed as in the case of acute toxicity

study. At the end of the experiment, all surviving animals were fasted overnight

before anesthetization with diethyl ether and sacrificed. The results were analysed

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.

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4.3.4.3. Clinical observations, mortality and body weight

All animals were examined for general behavior, signs of toxicity and

mortality, twice daily, as that in the case of acute toxicity study (section 4.3.3.3). The

BW of the rats were measured at the initiation of the experiment and then at weekly

intervals. The final BW of the rats was recorded following overnight fasting before

sacrifice. The gain in BW of all the tested rats recorded systematically.

4.3.4.4. Food and water consumptions

The feed consumptions per rat were recorded daily and at the termination of

the study, mean daily feed intake for each week was calculated. Throughout the study,

the water intake by the rats was also documented.

4.3.4.5. Viability of B. licheniformis Me1

Fresh faecal samples were collected from each treatment group along with the

control group rats on randomly selected days (10, 30, 50 and 80). This was done to

confirm if the administered test culture were able to survive the stress within the GIT.

The faeces were homogenized in normal saline (0.85% NaCl) and serially diluted.

The diluted homogenates (0.1 ml) were spread plated on LB agar plates for the

enumeration of B. licheniformis Me1. After incubation at 37ºC for 24 h, the numbers

of colonies were counted based on the colony appearance and recorded accordingly.

A control group of untreated rat was also analyzed.

4.3.4.6. Hematology

At the end of the experiment, all the surviving animals were fasted overnight

before anesthetization and blood samples were obtained by cardiac puncture from

anaesthetized animals for hematological analysis. Blood samples were collected in

two centrifuge tubes; one pre-filled with EDTA-2K as an anticoagulant and other

without any supplement. The hematological parameters which were determined with

an automated hematology analyzer, K-4500 (Sysmex Corp, Japan), included white

blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), mean

corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular

hemoglobin concentration (MCHC), platelet count (PLT) and differential leukocyte

count. For measuring the differential leukocyte count, blood samples were mixed with

1/4 volume of 5.0% EDTA-2K, and analyzed with a Microx HEG-120A (Omron

Tateishi Electronics Co., Ltd, Tokyo, Japan).

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4.3.4.7. Biochemical studies

The non-EDTA treated blood samples collected after sacrifice were used for

biochemical examination of the serum. The serum collected was used to determine the

levels of different metabolites and marker enzymes. The determination of urea,

creatinine, CK NAC, ALP, SGPT, SGOT and LDH, and the presence of Na+/K

+ and

Cl- ions were carried out using standard kits. The serum biochemical analysis was carried

out using the experimental protocols as described below. Blood biochemistry

determinations were performed manually using a spectrophotometer (Shimadzu, Japan).

4.3.4.7.1. Creatine kinase (CK NAC) assay

The CK NAC assay was performed as per the method described by Nielson

(1963) with further modification by the addition of EDTA. In 25 μl of sample, 1 ml of

reagent provided by the kit manufacturer (Aspen Laboratories Pvt. Ltd., India) was

added, mixed and incubated at 37ºC. After 2 min of incubation absorbance was

measured at 340 nm and again after 1 min and the mean absorbance change per min

(ΔA/min) was calculated. The CK NAC activity was expressed as,

CK (IU/L) = ΔA/min×TV×1000

d×ε×SV

Where, TV= Total reaction volume, 1000= Conversion of IU/ml to IU/L, d= Light

path in cm, ε = Millimolar absorptivity of NADH, SV= sample volume in ml.

4.3.4.7.2. Lactate dehydrogenase (LDH)

LDH was determined in rat serum following the method, as described

elsewhere (Henry et al. 1974). To the 25 μl of sample, 1 ml of reagent provided by the

kit manufacturer (Aspen Laboratories Pvt. Ltd., India) was added, mixed and

incubated at 37ºC for 60 sec. The absorbance of the resulting solution was measured

at 340 nm in 1 min interval and the change in absorbance (ΔA) was calculated. The

LDH was calculated using the formula,

LDH (IU/L) = ΔA/min×TV×1000

d× ε ×SV

Where, TV= Total reaction volume, 1000= Conversion of IU/ml to IU/L, d= Light

path in cm, ε = Millimolar absorptivity of NADH, SV= sample volume in ml.

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4.3.4.7.3. Determination of Creatinine

Quantitative determination of creatinine in plasma of rat was carried out

according to modified Jaffe’s method (Newman and Price 1999). To the 50 μl of

sample/standard, 1ml of working reagent provided by the kit manufacturer (Aspen

Laboratories Pvt. Ltd., India) was added and incubated at 37°C for 30 sec.

Absorbance, A1 was read at 505 nm and after 60 sec absorbance A2 was read. ΔA

(A2-A1) was calculated accordingly. The creatinine concentration was calculated as

follows,

Creatinine (mg/dl) = (ΔA of the sample) x Conc. of standard

ΔA of the Standard

4.3.4.7.4. Triglycerides

Determination of triglycerides was carried out using GPO-PAP, end point

assay (Stein and Myers 1995). To the 10 μl of standard/sample, 1 ml of reagent

provided by the kit manufacturer (Span Diagnostics Ltd., India) was added. The

reagent without the sample served as a blank. After incubation for 10 min at 37°C, the

absorbance was measured at 505 nm of standard and the test samples. The

triglycerides concentration was calculated as follows,

Triglycerides (mg/dL) = Absorbance of test× 200 (Dilution factor)

Absorbance of standard

4.3.4.7.5. Cholesterol

Total cholesterol (CHOD-PAP method) was determined in rat serum (Nader et

al. 1994). For the total cholesterol, 1 ml of reagent provided by the kit manufacturer

(Span Diagnostics Ltd., India) was added in aliquots of 10 μl standard/test samples

and incubated at 37°C for 10 min. The reagent without the standard/test sample

served as the blank. Absorbance of standard and test sample was measured at 505 nm.

The cholesterol concentration was calculated as follows,

Cholesterol concentration (mg/dL) = Absorbance of the test ×200 (Dilution factor)

Absorbance of standard

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4.3.4.7.6. HDL Cholesterol

High density lipid (HDL) cholesterol concentration was determined in two

steps using PEG-CHOD-PAP endpoint assay (Warnick et al. 1995). In step 1, 200 μl

of plasma was mixed with reagent 3 and incubated for 10 min, at room temperature.

The sample was centrifuged at 2000 g for 15 min and collected the clear supernatant

which was used for estimation. In step 2, reagent 1 provided by the kit manufacturer

(Span Diagnostics Ltd., India) was added to separate aliquots containing 100 μl of

supernatant and 100 μl of standard and incubated for 10 min at 37°C. Absorbance of

standard followed by test sample at 505 nm was measured. The HDL cholesterol was

calculated as follows;

HDL cholesterol (mg/dL) = Abs. of test × 50 × 2 (Dilution factor)

Abs. of Standard

4.3.4.7.7. Urea

Quantitative determination of urea was carried out by GLDH method (Burtis

and Ashwood 1999). To the 10 μl of sample/standard, 1 ml of working reagent

provided by the kit manufacturer (Aspen Laboratories Pvt. Ltd., India) was added.

After incubation for 30 sec at 37°C, ΔA (ΔA= A1-A2) was calculated by measuring

the absorbance A1 and A2 at 340 nm in 60 sec interval.

Urea (mg/dl) = ΔA of sample × Concentration of standard

ΔA of standard

4.3.4.7.8. Alkaline phosphatase

Modified DGKC method was used for the quantitative determination of

alkaline phosphatase in plasma (Burtis and Ashwood 1999). To the sample of 20 μl, 1

ml of working reagent provided by the kit manufacturer (Aspen Laboratories Pvt.

Ltd., India) was added and incubated for 1 min at 37°C. After incubation the

absorbance was read at 405 nm during 1 min interval. The ALP was determined using

the formula,

ALP= (ΔA/min) × 2750 (factor).

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4.3.4.7.9. Alkaline aminotransferase (SGPT) and Alanine aminotrasferase (SGOT)

Quantitative determination of SGPT and SGOT was done based on modified

IFCC method (Moss and Henderson 1999). To the 100 μl of sample, 1 ml of working

reagent provided by the kit manufacturer (Aspen Laboratories Pvt. Ltd., India) was

added and absorbance was measured at 340 nm. ΔA was measured according to the

absorbance difference at 1 min interval during incubation at 37 °C.

SGPT/ SGOT (U/L) = (ΔA/min) × 1745 (Factor)

4.3.4.7.10. Bilirubin

Quantitative measurement of total Bilirubin was determined in serum (Thomas

1998). To 100 μl samples, 50 µl each of reagent 1 and reagent 2, along with 1 ml of

reagent 3 was added. All the reagents were provided by the kit manufacturer (Aspen

Laboratories Pvt. Ltd., India). The blank consisted of only reagent 2 (100 μl) and

reagent 3 (1 ml), and 100 μl of sample. Absorbance of both the test and blank was

measured at 546 nm after incubation for 5 min at room temperature.

Bilirubin (mg/dl) = Abs. of the sample × Conc. Of the standard

Abs. of the standard

4.3.4.7.11. Glucose

Glucose concentration was determined using GOD-POD, endpoint assay

(Sacks 1999). To 10 μl each of sample and standard (400 mg/dL), 1 ml of working

glucose reagent provided by the kit manufacturer (Span Diagnostics Ltd., India) was

added. Blank was prepared by adding working reagent without sample. After

incubation at 37°C for 10 min, absorbance of the standard followed by test was

measured at 505 nm.

Plasma Glucose (mg/dL) = Abs. of the test ×100

Abs. of the Standard

4.3.4.7.12. Sodium assay

To 20 μl each of sample and sodium standard, 1 ml of precipitating reagent

provided by the kit manufacturer (Coral Clinical Systems, India) was added and

mixed for 5 min at room temperature. Clear supernatant was collected by

centrifugation at 3000 g. An aliquot of blank was prepared by adding acid reagent (1

ml), precipitating reagent (20 μl) and color reagent (100 μl). Standard and test was

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prepared by adding supernatant form step 1 instead of precipitating reagent in blank.

After mixing, the reaction preparation were kept for 5 min incubation at room

temperature and the absorbance of the blank (Abs. B), standard (Abs. S), and the test

sample (Abs. T) at 530 nm was measured against distilled water within 15 min. The

sodium assay was calculated as,

Sodium (millimol/L) = (Abs. B - Abs. T) × 150 (Factor)

(Abs. B - Abs. S)

4.3.4.7.13. Potassium assay

One milliliter of potassium reagent provided by the kit manufacturer (Coral

Clinical Systems, India) was added to separate tubes containing 20 μl of deionized

water, potassium standard and sample which served as blank, standard and test

respectively. After incubation for 5 min at room temperature, the absorbance was

measured at 630 nm of the blank (Abs. B), standard (Abs. S), and test sample (Abs. T)

against distilled water. The potassium assay was calculated as,

Potassium (millimol/L) = Abs. T × 5 (Factor)

Abs. S

4.3.4.7.14. Chloride assay

One milliliter of chloride reagent provided by the kit manufacturer (Coral

Clinical Systems, India) was added to separate aliquots containing 10 μl each of

deionized water, chloride standard and sample which served as the blank, standard

and test, respectively. Absorbance of the standard (Abs. S) and test (Abs. T) against

blank was measured at 505 nm after incubation for 2 min.

The chloride assay was calculated as,

Chloride (millimol/L) = Abs. T × 100 (Factor)

Abs. S

4.3.4.8. Relative organ weights and histopathological analysis

At the end of the treatment period, all the tested animals were sacrificed under

ether anesthesia. Gross observations were made at necropsy and recorded. At

necropsy, all the animals from experimental group and the control group were

anatomized for any lesions. Also, before further histopathological examinations, all

the organs/tissues were carefully examined macroscopically and the organ weights of

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liver, lungs, kidneys, heart, spleen, testis/ovary, epididymis/uterus, brain and adrenals

were noted. The relative organ weights were calculated based on the final BW of the

rats. At necropsy, the vital organs were surgically removed from the rats, washed with

normal saline, fixed and preserved in 10% neutral phosphate buffered formalin. All

the major tissues were further processed and trimmed, embedded into paraffin,

sectioned to a thickness of 4 µm and stained with hematoxylin and eosin (Bharucha et

al. 1976) and analysed by light microscopy. The collected tissues were grossly and

microscopically examined during histopathological examination.

4.3.5. Genotoxicity assay

4.3.5.1. Preparation of diet

The diet used for feeding mice was prepared according to Bureau of Indian

standards Specification for feed for laboratory animal – Part 1 (1970).

4.3.5.2. Micronucleus assay in mice

Animal care, handling and the environmental conditions in the experimental

room were as described before for rats (section 4.3.3.2). The animals (Swiss Albino

mice, Mus musculas, CFT strain) were maintained in pathogen-free facility and

housed in stainless steel cages. Adult male and female mice (Swiss Albino, Mus

musculas, CFT strain) weighing 36 ± 2 g were obtained from CFTRI animal house

facility. Animals were acclimatized to the experimental conditions for five days prior

to the start of the study. The study was conducted according to OECD Guideline for

the Testing of Chemicals No. 474; Mammalian micronucleus test, Adopted 21st July

1997. Mice of each gender were randomly assigned into four groups and housed (5

animals in a single cage). The animals were weighed and observed for signs of illness

or other abnormalities at the start of the study. Ethane methane sulphonate (EMS) (5

μg/ml) and water were used as positive and negative controls, respectively. The test

culture, B. licheniformis Me1 was administrated to two different mice groups at two

concentrations, 1.1×1010

CFU/kg BW and 1.1×1011

CFU/kg BW of mice, using water

as a vehicle. The test culture was given by oral route for two consecutive days at 24 h

interval. The positive and negative controls were given to the control groups mice

only once by intraperitoneal injection on the last day of test culture administration.

After 36 h of the last day of dose administration, blood samples from the mice were

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174 Nithya V.

collected by tail trimming. Peripheral blood samples were smeared on acridine orange

coded slides and observed under florescent microscope. At least 2,000 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.

4.3.6. In vivo safety assessment in rabbits

4.3.6.1. Animals and husbandry

Male albino rabbits, weighing 2500 ± 250 g from CFTRI animal house facility

were used for the study. Animals were housed individually in metal cages at 20 ±

3°C, relative humidity of 40-70% 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.

4.3.6.2. Acute eye irritation study in rabbits

The dosage for the study was 0.1 g of the undiluted cell mass containing

1.93×1011

CFU/g. Detailed clinical observations were made prior to exposure of the

animals to the test culture. Three healthy male albino rabbits were used for acute eye

irritation study. Both eyes of the animals were examined 24 h prior to the start of the

study. Only animals not showing any ocular defects or pre-existing corneal injury

were used in the experiment. The dosage of test bacterial culture (0.1 g of the

undiluted cell mass at a concentration of 1.1×1013

CFU/g) was placed into the

conjunctival sac of the left eye of a single rabbit. After determination that the pain

reaction was very low or negligible and that anesthesia was not required with

application of the test culture, the dose was added to the eyes of the remaining two

animals. The untreated right eye served as control. Eyes of the rabbit were not washed

after the application. Eyes were examined at 1, 24, 48 and 72 h after the test culture

application for any eye lesions or treatment-related severity and nature or duration of

reactions. Any clinical signs of toxicity or signs of ill-health of the animals were

recorded during the study. At the end of 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.

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175 Nithya V.

4.3.6.3. Acute skin irritation study in rabbits

Three healthy male albino rabbits were used for acute skin irritation study. An

undiluted dose of 0.5 g test culture (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 three animals and covered

with sterile gauze patches, held in place with non irritating tape. The trunks of the

animals were wrapped in plastic wrap for 4 h, which was the duration of the test item

exposure. After 4 h of exposure, the test culture was removed from the animal’s skin

by washing with water. Untreated skin areas of each animal served as control.

Animals were examined for erythema and edema at 1, 24, 48 and 72 h after the test

culture removal. The test culture was evaluated according to the Draize (1977)

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.

4.3.7. Statistical analysis

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 separately, were carried out by one-way analysis of variance

(ANOVA), and a post-hoc analysis of individual pair difference was performed by

Duncan’s multiple range tests. All the data are presented as means ± SEM. A p-value

of < 0.05 was taken as statistically significant.

4.4. Results

4.4.1. Acute oral toxicity study

A fourteen-day oral acute toxicity study in adult male albino Wistar rats was

performed to investigate the effect of test culture, B. licheniformis Me1

administration. These results provides 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 clinical related signs and deaths were observed during acute toxicity

studies in the treated male rats during 14 consecutive days. A single dose of 1.1×1011

CFU/kg BW of rat did not show any treatment-related signs in behavioural pattern,

changes in locomotors activity, respiratory, digestive, circulatory, autonomic activity

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176 Nithya V.

and the central nervous system. No obvious signs of toxicity, no abnormality in skin,

fur and eyes, and also no changes in other physiological activities immediately after

giving dose or during post treatment period in any of the animals were observed. Feed

and water uptake was normal, and there was no loss or gain in BW of the rats 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 organism was found to be safe at the tested dose level as per OECD guidelines.

4.4.2. Subchronic oral toxicity in rats

4.4.2.1. Mortality and clinical symptoms

A 13-week repeated-dose toxicity study was performed in rats to determine a

NOAEL for defined toxicological endpoints and was used to establish a safe chronic

oral dose for humans. During this experimental period, all the animals survived from

the test culture administration. Animals of both the groups appeared and behaved

normal in their cages throughout the experimental period, with no clinical signs of

toxicity or allergic reactions. No treatment related incidence of diarrhoea, constipation

or other gastrointestinal disorders and changes in locomotors activity, respiratory,

circulatory, autonomic and central nervous system were observed. On necropsy,

macroscopic observation of the test and control group animals revealed no alterations

in the external surfaces of the organs and all the orifices of the cranial, thoracic and

abdominal cavities were normal.

4.4.2.2. Feed and water consumption, body weight

Table 3 shows the daily feed intake of male and female rats over 13 weeks.

Male rat group fed with a test dose of 1.1×1011

CFU/kg BW of rat, showed a

significantly higher (p<0.05) feed intake at the end of the experimental period. In all

other animal groups, feed intake was normal for both male and female rats without any

significant difference between the control and experimental groups, respectively,

throughout the experiment (Table 4.3). During the entire experimental period, the water

intake was normal for all the rat groups. Also, throughout the study, no statistically

significant changes concerning the BWs were noticed in test culture fed groups in male

and female Wistar rats when compared with control groups (Fig. 4.1 and Fig. 4.2).

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177 Nithya V.

4.4.2.3. Viability of B. licheniformis Me1

Viability of the test culture in the rat GIT was determined by monitoring the

presence of B. licheniformis Me1 in the faeces of treated rats during the experimental

period (day 10, 30, 50 and 80). An average of 1×106 CFU of B. licheniformis Me1

per gram of the rat’s faeces was observed. There was no marked difference in the

numbers of B. licheniformis in the faeces across test groups on any of the days tested.

From this study, it can be concluded that B. licheniformis Me1 was able to survive the

GIT conditions and not all fed test culture was excreted out.

Figure 4.1. Graph showing body weight of male Wistar rats given test article for 13 weeks.

Control ( ), 1.1x1010

CFU/kg BW (), 1.1x1011

CFU/kg BW (). Values are

means ± SEM of six rats,

Figure 4.2. Graph showing body weight of female Wistar rats given test article for 13

weeks. Control ( ), 1.1x1010

CFU/kg BW (), 1.1x1011

CFU/kg BW ().

Values are means ± SEM of six rats.

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10 11 12 13

Bo

dy

we

igh

t (g

)

Week

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10 11 12 13

Bo

dy

wei

ght

(g)

Week

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178 Nithya V.

Table 4.3. Daily feed intake of male and female Wistar rats fed with test article for 13 weeks

Daily feed intake (g)

Week Male Female

Control 1.1×1010

CFU/kg BW 1.1×1011

CFU/kg BW Control 1.1×1010

CFU/kg BW 1.1×1011

CFU/kg BW

1 10.26 ± 0.52 10.03 ± 0.32 10.30 ± 0.37 10.66 ± 0.42 10.66 ± 0.50 10.76 ± 0.48

2 12.55 ± 1.18 11.98 ± 1.08 12.72 ± 0.63 12.04 ± 0.44 11.97 ± 0.58 12.15 ± 0.81

3. 14.41 ± 2.1 12.89 ± 1.43 13.79 ± 0.66 12.24 ± 1.43 12.06 ± 1.19 12.27 ± 1.77

4. 14.03 ± 2.17 13.10 ± 1.19 13.98 ± 0.54 12.59 ± 0.98 12.83 ± 0.87 12.41 ± 1.47

5. 14.06 ± 0.87 13.71 ±1.38 15.23± 1.26 12.96 ± 1.21 12.56 ± 1.36 12.52 ± 1.21

6. 14.39 ± 0.14 14.32 ±1.71 15.32 ± 1.06 13.07 ± 0.67 12.72 ± 2.36 12.77 ± 1.61

7. 14.37 ± 0.59 14.66 ±1.09 15.12 ± 1.44 13.6 ± 0.88 12.96 ± 2.38 12.96 ± 1.73

8. 14.07 ± 0.32a 14.92 ±1.3

a 16.20 ± 1.00

b 14.09 ± 2.24 13.28 ± 0.70 13.12 ± 1.60

9. 15.51 ± 0.41 15.28 ± 1.27 16.50 ± 1.24 14.29 ±1.77 14.10 ± 1.34 13.54 ± 1.53

10. 16.03 ± 0.52a 15.98 ± 0.92

a 16.67 ± 0.82

b 14.64 ± 1.12 14.34 ± 1.93 13.86 ± 0.89

11. 16.06 ± 0.80 16.18 ± 1.2 16.81 ± 0.34 14.93 ± 0.25 14.54 ± 2.09 14.17 ± 1.06

12. 16.15 ± 0.73 16.49 ± 1.70 17.14 ± 0.90 15.07 ± 0.62 14.89 ± 1.77 14.34 ± 1.15

13. 16.52 ± 0.58 16.84 ± 1.86 17.89 ± 0.99 15.15 ± 1.34 15.17 ± 2.08 15.17 ± 0.99

Values are means ± SEM, n = 6. Values in the same row in male and female group that do not share the same alphabetic superscripts are significantly different at 5% levels

according to Duncan’s multiple range test.

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179 Nithya V.

4.4.2.4. Relative organ weights

At necropsy, macroscopic observation of the organs revealed no treatment

related damages or differences. The relative organ weights of adrenals, spleen, kidney

and heart did not show any significant deviation from that of the control in any of the

treated animal groups (Table 4.4, Table 4.5). However, a marginal but statistically

non significant decrease in relative organ weight of lungs in females was noticed

(Table 4.5). A dose dependent reduction of kidney fat, adipose fat and testicular

fat/ovular fat was observed in tested groups of males and females when compared to

the control groups. However, these effects were only significant for male’s kidney and

testicular fat.

4.4.2.5. Histopathology

Histopathological examination of the tissues of control as well as treated

groups revealed no treatment related abnormalities in morphology or toxicity to the

organs. Livers of the treated animals showed no shrunken hepatocytes or congestions

in portal tracts and sinusoids. Sections showed well formed structures of parenchyma

and portal triads. Lungs showed organized alveolar spaces and also no thickening of

inter alveolar septa or cellular infiltrations were observed. Kidneys of treated animals

were also normal and did not display any glomerular or vascular congestion. No

swelling of epithelium and occlusion of lumen were noticed even in the highest dose

group. The villi pattern of the small intestine was well preserved in all the rats fed

with B. licheniformis Me1. Other vital organs, like heart and brain also showed

normal structure. Histological examination of the ovaries of the treated groups

revealed different stages of follicular development. No abnormalities were observed

in germinal epithelium, stages of follicular development, maturation and corpus

luteum. There were no microscopic or macroscopic lesions in any organs that could

be attributed due to the treatments. In this study, histopathological investigations

failed to reveal any incidence of organ toxicity.

4.4.2.6. Haematology and serum biochemical studies

No significant differences were found in any of the analysed aspects in

haematology for both male and female test groups when compared to that of the

control groups (Table 4.6). Also, on serum biochemical analysis, no statistically

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180 Nithya V.

significant dose dependent alterations were detected either in the levels of glucose,

cholesterol, triglyceride, urea, or in the activity of ALP, LDH, AST and ALT in both

the sexes in comparison to the control groups (Table 4.7).

Table 4.4. Relative organ weights of male Wistar rats fed with the test organism for

13 weeks

Organ (g %) Control 1.1×1010

CFU/kg BW 1.1×1011

CFU/kg BW

Brain 0.64 ± 0.06 0.63 ± 0.04 0.63 ± 0.06

Liver 0.55 ± 0.09 0.55 ± 0.09 0.54 ± 0.08

Heart 0.32 ± 0.02 0.32 ± 0.05 0.33 ± 0.02

Lungs 3.05 ± 0.24 3.14 ± 0.24 3.11 ± 0.20

Spleen 0.21 ± 0.01 0.21 ± 0.05 0.22 ± 0.05

Kidney 0.76 ± 0.09 0.75 ± 0.05 0.75 ± 0.05

Adrenal 0.03 ± 0.02 0.03 ± 0.03 0.03 ± 0.01

Testis 1.09 ± 0.07 1.05 ± 0.16 1.07 ± 0.15

Epididymis 0.31 ± 0.02 0.29 ± 0.03 0.30 ± 0.02

Thymus 0.075 ± 0.07 0.081 ± 0.03 0.076 ± 0.01

Values are means ± SEM, n ± 6. There was no significant difference between groups at 5% levels

according to Duncan’s multiple range tests.

Table 4.5. Relative organ weights of female Wistar rats fed with the test organism

for 13 weeks

Organ (g %) Control 1.1×1010

CFU/kg BW 1.1×1011

CFU/kg BW

Brain 0.73 ± 0.06 0.75 ± 0.06 0.76 ± 0.09

Liver 0.59 ± 0.06 0.58 ± 0.06 0.59 ± 0.05

Heart 0.36 ± 0.05 0.35 ± 0.04 0.36 ± 0.04

Lungs 3.13 ± 0.27 2.92 ± 0.11 2.91 ± 0.11

Spleen 0.22 ± 0.01 0.23 ± 0.04 0.22 ± 0.05

Kidney 0.81 ± 0.08 0.81 ± 0.10 0.80 ± 0.07

Adrenal 0.03 ± 0.01 0.04 ± 0.00 0.04 ± 0.02

Ovaries 0.06 ± 0.01 0.05 ± 0.01 0.06 ± 0.01

Uterus 0.321 ± 0.02 0.308 ± 0.01 0.319 ± 0.02

Thymus 0.086 ± 0.05 0.089 ± 0.02 0.091 ± 0.01

Values are means ± SEM, n ± 6. There was no significant difference between groups at 5% levels

according to Duncan’s multiple range tests.

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181 Nithya V.

Table 4.6. Hematological analysis for male and female Wistar rats fed with the test article for 13 weeks

Parameter Male Female

Control 1.1×1010

CFU/kg BW 1.1×1011


CFU/kg BW 1.1×1011

CFU/kg BW

WBC (103/μl) 14.26 ± 4.37 14.27 ± 4.00 14.76 ± 4.10 ±

RBC (106/μl) 9.10 ± 0.55 9.44 ± 0.20 9.03 ± 0.29

HGB (g/dL) 15.13± 0.73 14.90 ± 0.35 15.23 ± 0.11

HCT (%) 50.87 ± 1.95 50.50 ± 1.67 51.33 ± 0.05

MCV(fL) 55.97 ± 2.48 56.23 ± 1.36 56.87 ± 1.81

MCH (pg) 16.63 ± 0.20 16.93 ± 0.46 16.87 ± 0.65

MCHC (g/dL) 29.73 ± 0.92 30.13 ± 0.49 29.67 ± 0.23

PLT (103/μl) 919.67 ± 48.04 902.67 ± 35.21 906.00 ± 59.25

Differential count (%)

N 14.33 ± 0.50 15.00 ± 1.36 14.33 ± 0.93

L 81.66 ± 0.80 81.67 ± 0.51 81.33 ± 0.69

E 1.50 ± 0.50 1.33 ± 0.57 1.67 ± 0.58 .60

M 2.50 ± 0.51 2.42 ± 0.51 2.67 ± 1.53

B - - - - - -

Values are means ± SEM, n ± 6. There was no significant difference between groups at 5% levels according to Duncan’s multiple range tests.

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182 Nithya V.

Table 4.7. Biochemical analysis for male and female Wistar rats fed with test article for 13 weeks

Parameter Male Female

Control 1.1×1010

CFU/kg BW 1.1×1011


CFU/kg BW 1.1×1011

CFU/kg BW

Creatinine (mg/dL) 0.78 ± 0.05 0.79 ± 0.16 0.73 ± 0.09 0.80 ± 0.03 0.83 ± 0.45 0.81 ± 0.13

Urea (mg/dL) 41.39 ± 3.39 40.06 ± 4.63 39.4 ± 3.26 40.49 ± 0.03 41.84 ± 0.45 40.30 ± 0.13

Total cholestrol (mg/dL) 83.29 ± 4.01 85.09 ± 5.35 80.77 ± 3.25 77.28 ± 0.39 75.97 ± 0.77 76.38 ± 0.63

HDL cholesterol (mg/dL) 56.69 ± 2.14 52.32 ± 9.44 53.97 ± 7.31 55.12 ± 0.74 54.55 ± 1.76 52.78 ± 0.57

Triglycerides (mg/dL) 80.90 ± 1.53 82.90 ± 0.91 83.51 ± 0.78 69.18 ± 1.46 70.21 ± 4.99 71.45 ± 1.79

Glucose (mg/dL) 70.86 ± 1.56 69.22 ± 8.90 70.17 ± 0.55 69.08 ± 8.37 63.02 ± 4.79 64.47 ± 9.79

ALP (U/L) 250.25 ± 59.75 257.23 ± 87.35 250.79 ± 81.71 158.1 ± 91.36 177.45 ± 46.62 140.35 ± 37.68

SGOT (U/L) 289.18 ± 6.49 284.87 ± 10.69 289.23 ± 6.25 352.49 ± 9.23 366.83 ± 5.49 358.63 ± 4.15

SGPT (U/L) 50.61 ± 7.71 49.11 ± 6.83 51.92 ± 3.24 56.10 ± 4.40 56.69 ± 5.20 55.45 ± 3.32

CK NAC (IU/L) 2664.41 ± 28.94 2699.43 ± 51.27 2722.44 ± 14.34 2177.47 ± 37.29 2083.07 ± 55.93 2135.0 ± 68.28

LDH (IU/L) 3162.75 ± 55.75 3021.32 ± 41.74 3129.56 ± 34.69 1458.39 ± 47.04 1401.66 ± 89.9 1404.21 ± 80.22

Total bilirubin (mg/dL) 18.75 ± 0.00 17.67 ± 2.92 17.74 ± 1.52 10.20 ± 0.79 9.63 ± 1.48 10.52 ± 0.00

Sodium (mmol/l) 141.75 ± 13.79 139.00 ± 15.17 146.00 ± 6.76 150.50 ± 8.49 148.00 ± 13.94 145.00 ± 0.00

Potassium (mmol/l) 5.27 ± 0.52 4.41 ± 1.01 4.01 ± 1.27 5.23 ± 0.61 5.18 ± 0.30 5.52 ± 0.43

Calcium (mmol/l) 84.02 ± 6.5 81.33 ± 4.03 82.24 ± 7.77 92.27 ± 6.56 97.48 ± 6.88 95.19 ± 3.89

Values are means ± SEM, n = 6. There was no significant difference between groups at 5% levels according to Duncan’s multiple range tests.

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183 Nithya V.

4.4.3. Micronucleus assay in mice

The micronucleus test was conducted to investigate the formation of

micronuclei containing chromosome fragments or whole chromosomes and is

considered as 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 culture 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. None of the treatment groups were positive for statistically significant

induction of micronuclei in reticulocytes, and the ratio of reticulocytes to total

erythrocytes in these groups showed no significant decrease compared to the negative

control group. The ratio of reticulocytes to total erythrocytes between the treated

animals also did not show any significant difference. For female mice, the average

reticulocytes to total erythrocytes ratio in the negative control group was 2.17%. Also,

the treated groups 1.1×1011

and 1.1×1010

CFU/kg BW/day showed 2.07% and 1.93%

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 two dose groups 1.1×1011

and

1.1×1010

CFU/kg BW/day exhibited 1.51% and 1.10% reticulocytes to erythrocytes

ratio respectively. In the male group, the positive control showed 27.12% decrease in

the ratio. These results clearly show that the test culture did not cause any signs of

bone marrow cytotoxicity of mice in the range of test doses.

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 (Endres et al

2009). 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 culture dose

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184 Nithya V.

levels of 1.1×1011

and 1.1×1010

CFU/kg BW/day, respectively. Since, the mice

peripheral blood micronucleus assay did not show any statistically significant changes

according to the OECD guidelines, there is no indication that the test culture B.

licheniformis Me1 administration caused any genocytotoxicity.

4.4.4. Safety studies in rabbits

4.4.4.1. Acute eye irritation study in rabbits

The test culture B. licheniformis Me1 application to the mucosa of the eyes did

not result in any conjunctival irritant effect neither after 1 h nor later during the

experimental period. Also, any negative symptoms in either the cornea or the iris were

observed. According to the European Commission (EC) criteria of 2001/59/EEC for the

classification and labeling requirements for dangerous substances and preparations, the

test culture is not required to be classified or labeled as irritant to the eye.

4.4.4.2. Skin irritation study in rabbits

The application of B. licheniformis Me1 cell mass to the skin did not reveal

any clinical signs of erythema and edema at 1 h after removal of the patch and until

72 h later. According to the EC directive 2001/59/EEC, it is therefore not required to

classify or label the test culture as skin irritant.

4.5. Discussion

As the demand for health promoting and minimally processed functional foods

among consumers continues to grow in the future, several new foods are likely to

include probiotics and use the ABP from microbes for biopreservation of food

products. Many reports have shown that selected strains of Bacillus, with a history for

safe use in the food industry, are increasingly being incorporated in health promoting

“functional foods” to provide digestive and immune health benefits (Durkee 2012;

Hosoi and Kiuchi 2003; Pinchuk et al. 2001; Samanya and Yamauchi 2002) and

control the growth of spoilage microorganisms in food products (Bizani et al. 2008;

Martirani et al. 2002). The ability of B. licheniformis to produce a wide array of

antimicrobial substances with a broad inhibitory spectrum (Abriouel et al. 2010) and

promising probiotics characteristics (Bilev 2002; Sorokulova 1997)

makes it a suitable

and promising candidate strain for application in probiotic food products and its

metabolite, ABP for biopreservation of foods. However, it is of obvious importance

that novel cultures and its metabolites for human and animal consumption are

evaluated carefully and precisely for safety and efficacy before commercialization.

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185 Nithya V.

Studies, including acute (single administration) toxicity and a repeated

administration (chronic) toxicity assessment have been recommended for assessing

the safety of cultures (FAO/WHO 2002; Ishibashi and Yamazaki 2001). To assess the

safety of B. licheniformis Me1 strain, an in vivo acute and subchronic toxicity study

was conducted. In acute toxicity study, a limit test was conducted with a maximum

dose level of 1.1×1011

CFU/kg BW of rat, and this administered dose resulted in no

treatment-related mortality or illness. This result indicated that the oral LD50 for the

culture was ≥1.1×1011

CFU/kg BW of rat. Since, no treatment-related mortality,

morbidity or clinical symptoms resulted in this acute oral toxicity study using a single

dose of 1.1×1011

CFU/kg BW of rat, B. licheniformis Me1 can be considered nontoxic

as per the OECD guidelines. Similar results were reported for the safety analysis of

other probiotic strains of Bacillus spp., such as B. subtilis and B. licheniformis

(Sorokulova et al. 2008) and B. coagulans (Endres et al. 2009).

Organ weight changes have long been accepted as a sensitive indicator of

chemically induced changes to organs. Therefore, in toxicological experiments, the

comparison of organ weights between control and treated groups have conventionally

been used to predict the toxic effect of a test culture (Peters et al. 1966). The absence

of significant changes in the vital organs of the treated groups during the subchronic

oral toxicity study shows that the ingestion of B. licheniformis Me1 did not induce

any anomalous lesions or inflammation of these organs. Similar observations were

found by Sorokulova et al. (1997) in a safety assessment of probiotic Biosporin,

whereas, administration of B. coagulans resulted in difference of organ weight of

liver, brain and hemorrhages in the lungs (Endres et al. 2009) The reduction in fat

deposition observed in the rat groups, fed with the test culture can be correlated to the

health benefits of the culture B. licheniformis Me1.

Hematological values were not significantly affected by fortification of B.

licheniformis Me1 between the control and the treated groups for all the parameters

analyzed. Hong et al. (2008) showed similar findings in toxicity studies of B. subtilis

and B. indicus conducted in rabbits. Results of the present study suggest that the test

culture, B. licheniformis Me1 may not be toxic as they do not significantly affect the

circulating red blood cells nor the haematopoiesis or leucopoiesis that could otherwise

have caused a megaloblastic anaemia, or significant changes in packed cell volume

(PCV) and eosinophils. Furthermore, the normal metabolism of the treated animals

was not affected by the test culture administration.

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186 Nithya V.

There were no significant alterations in liver function parameters investigated

in this study, including total protein, bilirubin and the activity of liver enzymes,

including LDH, ALT and AST. Also, there were no gross or microscopic pathological

changes found in the liver. Absence of any elevated activity of diagnostic marker

enzymes and no significant toxicological impact suggests the safety of test culture

augmentation at levels used in the present study. Absence of any significant

histological findings viz. cellular infiltrations, inflammation and lesions in the vital

organs emphasize the safety aspect of B. licheniformis Me1 at levels given in this

experiment. Results obtained are in accordance with the data for Lactobacillus (Zhou

et al. 2000) and Bacillus (Endres et al. 2009) probiotics.

The viability test results showed that not the entire fed test culture, B.

licheniformis Me1 was excreted out in the faeces of the rats. This shows the viability

of the test culture within the rat GIT and also shows that the ingested dose actually

reaches the region of the GIT where it should exert its effect (Hamilton-Miller and

Gibson 1999). Having reached the small intestine, a proportion of the ingested test

culture would have colonized in the nutrient rich region and would have established

itself alone or in association with other gut microbes in the small intestine. Several

other authors have also reported the persistence of Bacillus spp. in the GIT of the

experimental animal models (Casula and Cutting 2002; Duc et al. 2004; Tam et al.

2006). SCAN (2002) reported the presence of approximately 80% of the fed B.

licheniformis NCTC13123 in a vegetative form in the pig digestive tract by doing a

microbiological examination of faeces collected from piglets during a tolerance test.

The in vivo micronucleus test conducted in mice to assay the cytogenotoxic effect of

B. licheniformis Me1 showed that there was no statistically significant dose-related

increase in the incidence of micronucleated immature erythrocytes for the treatment

groups compared with the concurrent control group. Also, there were no substantial

and statistically significant dose-related decreases in the proportion of immature

erythrocytes. These findings were similar to that observed for B. coagulans (Endres et

al. 2009). Acute eye and skin irritation tests conducted in rabbits showed no signs of

eye irritation and skin erythema or edema. A similar result for skin irritation test was

reported on application of B. licheniformis NCTC13123, while in eye irritation study,

a diffuse corneal opacification and evidence of conjunctivitis were observed (SCAN

Chapter – 4


187 Nithya V.

2002). Endres et al. (2009) also reported a slight conjunctiva irritation and erythema

on the treated skin surface within one hour of B. coagulans application. However,

they reported the reaction was fully reversible later and thus, it can be assumed that

these reactions might be due to the technical manipulation of administration.

The concentration of B. licheniformis Me1 (1.1×1011

CFUs/kg BW of rat)

used in this study corresponds to 77×1011

CFUs for an average 70 kg human being.

Thus, the concentration used can be considered 2566 to 77,000 times safe for human

consumption, as the suggested human dose is in the range of 1×108 to 3×10

9 CFUs.

4.6. Conclusion

The native isolate from food B. licheniformis Me1 administered to rats at a

dose of 1.1×1011

CFU/kg BW did not show any sign of toxicity or mortality in in vivo

oral acute and subchronic assessments. Hence, the NOAEL for both males and

females is considered to be higher than 1.1×1011

CFU/kg BW of rat per day, which

was the highest dose tested. Furthermore, a micronucleus assay carried out in mice

using B. licheniformis Me1 did not demonstrate any evidence for cytogenotoxicity. In

addition, applications of B. licheniformis Me1 did not produce any signs of skin or

eye irritation. Based on these results and according to the highest dose levels required

by OECD guidelines for materials of low toxicity, it can be concluded that the culture

B. licheniformis Me1 is safe in rodents and can be considered safe for use in food

industry either as biopreservative or as probiotics.

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In vivo safety assessment of bacteriocinogenic Bacillus licheniformis Me1shodhganga.inflibnet.ac.in/bitstream/10603/17837/9/14... · 2018-07-09 · Bacillus licheniformis Me1 performed