evaluation of microbiological and physicochemical …

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839

Himanshu et al. World Journal of Pharmacy and Pharmaceutical Sciences

EVALUATION OF MICROBIOLOGICAL AND PHYSICOCHEMICAL

PROFILE OF PROBIOTIC LACTOBACILLUS SPOROGENES AS A

BIOTHERAPEUTICS AGENT

Himanshu K. Solanki1*

and Dushyant A. Shah2

1Department of Pharmaceutics, S.S.R. College of Pharmacy, Sayli-Silvassa Road, Sayli,

Silvassa, U.T. of D.N.H.-396230, India.

1Ph.D Scholar, Hemchandracharya North Gujarat University, Patan-384265, Gujarat, India.

2APMC College of Pharmaceutical Education and Research, Motipura, Himmatnagar-

383001, Dist: Sabarkantha.India.

ABSTRACT

Introduction: The aim of the present study was to evaluate

microbiological and physicochemical profile of probiotic Lactobacillus

sporogenes spores to aid designing of stable formulations. The

probiotic are susceptible to loss in viability due to formulation,

processing, storage and in vivo environment. Lactobacillus

sporogenes a revolutionary new friendly bacteria naturally occurring in

the intestine. It is a spore-forming bacterium that makes it the choice

of probiotic with enormous clinical applications. Methods:

Lactobacillus sporogenes spores were studied for microbiological

studies, organoleptic properties, Microscopic examination, Scanning

electron microscopy (SEM), X-ray diffraction study, Grams staining,

catalase test, Bile salt tolerance, Quantitative test for lactic acid

Production, Microbial limit test, Loss on Drying, aqueous pH stability, flow properties and

excipient compatibility. Result and Conclusions: Aqueous suspension of Lactobacillus

sporogeness in buffer solutions of pH 1.2 to 8 showed rapid degradation with maximal

stablility in intestinal pH 6.8. The spores were found to be compatible with the excipients

evaluated, with noted exception of Sodium alginate, HPMC K4M, Sodium CMC, Carbopol

934 P. The physicochemical profiling of L. sporogenes presented in the study provides

understanding of the material attributes critical to product design in terms of selection of

formulation ingredients, process conditions and pack suitability.

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Article Received on

20 Dec 2014,

Revised on 13 Jan 2015,

Accepted on 07 Feb 2015

*Correspondence for

Author

Himanshu K. Solanki

Department of

Pharmaceutics, S.S.R.

College of Pharmacy,

Sayli-Silvassa Road,

Sayli, Silvassa, U.T. of

D.N.H.-396230, India.


840


KEYWORDS: Probiotic, Lactobacillus sporogenes, Characterization, Stability,

Biotherapeutics Agent.

INTRODUCTION

The word “probiotic” literally means “good life,” and the real world applications are very

much in line with that definition. Probiotics are live microbial feed supplements that can

benefit the host by maintaining the balance of intestinal microflora [1]

.

Probiotics, as defined by the Food and Agricultural organization (FAO) of the United Nations

and the World Health Organization (WHO) are “live microorganisms (bacteria or yeasts),

which when ingested or locally applied in sufficient numbers confer one or more specified

demonstrated health benefits for the host” [2,3]

.

Preformulation testing is the first step in the rational development of dosage forms of the

drug. It can be defined as an investigation of physical and chemical properties of drug

substance, alone and when combined with excipients.

The overall objective of preformulation testing is to generate information useful to the

formulator in developing stable and bioavailable dosage forms, which can be mass-produced.

A thorough understanding of physicochemical properties may ultimately provide a rationale

for formulation design or support the need for molecular modification or merely confirm that

there are no significant barriers to the compound development.

The goals of the program therefore are.

To establish the necessary physicochemical characteristic of a new drug substance.

To determine its kinetic release rate profiles.

To establish its compatibility with different excipients.

Here, preformulation studies on the obtained sample of Lactobacillus sporogenes include

physical tests and compatibility studies [4, 5]

.

B. coagulans commonly mislabeled as Lactobacillus sporogenes, [6]

has a long history of use

as a probiotic. L. sporogenes unique among probiotics in that it possesses a protecting, spore-

like protein covering, which allows it to survive stomach acid, arrive at the small intestine,

germinate, and grow [7-9]

.


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Recently one strain of B. coagulans, labelled as GanedenBC30 has been approved self-

affirmed GRAS (Generally Recognized as Safe) category by the FDA [6, 9]

in the US.

Marketed by Ganeden, as GanedenBC30 it is being used in a number of products such as

Sustenex and is also being included into foods where spores can survive the gentle heat-

treatments used to sterilize foods.

Apart from dietary supplement, bacillus probiotics are used as a therapeutic product for the

treatment of gastrointestinal and urinary tract infections. The therapeutic benefit is partly due

to the ability of L. sporogenes to secrete a bacteriocin, coagulin, which is active against a

broad spectrum of enteric microbes [10]

.

Like other probiotic strains it also suffers wide variation between the actual content and the

labeled claim of viable spores. To compensate for this, very high amount of overages are

added, which not only increase the cost of production but also result in variable dose of the

probiotic.

Problems in the stability of microorganisms commonly used in food industry have been

documented [11]

.

Although these studies were not carried out strictly as per the International Conference on

Harmonization (ICH) guidelines for stability testing, they indicated significant fall in the

probiotic content upon storage. Probiotics have been reported to be susceptible to pH

conditions and moisture [12, 13]

.

The effect of external stress parameters on L. sporogenes can yield information on its

formulation requirements and storage conditions. Thus, the particular aims of this research

effort were (1) to determine the aqueous pH on viability of L. sporogenes; and (2) to

determine the compatibility of L. sporogenes with commonly used pharmaceutical excipients.

MATERIAL AND METHODS

Materials

Lactobacillus sporogeness from Unique Biotech Ltd., Hyderabad, India, MRS Agar, Glucose

yeast extract agar (GYE) agar medium, Nutrient Agar Media, from Hi-media, Mumbai, India.

Hydroxypropyl methylcellulose (HPMC) and Carbopol were procured from Colorcon Asia

Pvt. Ltd. (Mumbai, India), Sodium alginate were procured from Chemdyes Pvt. Ltd, Rajkot,


842


India, Sodium Carboxy Methyl Cellulose were procured from Astron Chemicals,

Ahmedabad, India.

Experimental Methods

Different selective growth medium

The growth of Lactobacillus sporogeness in different selective medium namely, MRS agar

[14], Glucose yeast extract agar media and Nutrient agar was evaluated. 100 µL of

Lactobacillus sporogenes suspension was spread on the plate of each medium and incubated

at 37C for 48 hours in B.O.D Incubator [Bio-tech (BTI-06) India]. The colonies were

isolated and examined in terms of morphology like size, shape colour of colony and using

gram staining. The viable count of Lactobacillus sporogenes in all the media was also

obtained and compared using the colony counter [Chemline (CL-910), India].

Effect of inoculation method

The viable counts of Lactobacillus sporogenes bacteria were obtained in GYE Agar Media

using two different methods namely pour plate and spread plate method. The two methods

were compared to find out suitable method for counting the colony of Lactobacillus

sporogenes both methods were conducted by ten fold serial dilution of the original culture.

The pour plate method was applied by transferring 1 ml from 105 diluted Lactobacillus

sporogenes into a sterile petri-dish and 20 ml of GYE Agar Media were then poured into the

petri-dish. The plate was left for approximately 30 min to solidify and kept in an incubator at

37 C up to 24 hours.

On other hand, the spread plate method was conducted by adding 0.1 ml from 105 dilution

onto the surface of the GYE Agar Media plates and spread using a sterile spreader. The

number of colony was counted using a colony counter.

The experiments were repeated in triplicate.

Organoleptic Characteristics

The color, odor, and taste of the Lactobacillus sporogeness were characterized and recorded

using descriptive terminology.


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Microscopic Examination

Small quantity of Bacilluscoagulanspowder was mounted on glass slides by using spatula.

cover with a cover slip. Examine using a microscope (Microtech, India).

Surface Morphology using Scanning electron microscopy (SEM)

The shape and surface topographical characteristics properties Lactobacillus sporogenes

powder were investigated by scanning electron microscopy (SEM) ZEISS LS10 EVOSEM at

Tata institute of fundamental Research, Navvy nagar, Colaba, Mumbai.

Lactobacillus sporogenes powder were platinum coated by mounted on a stub using double-

sided adhesive tape and under vacuum in an auto fine coater (ZEISS LS10 EVOSEM) to

make them electrically conductive and their morphology was examined by SEM at different

magnification.

X-ray diffraction study

X-ray powder diffractograms of Lactobacillus sporogenes were recorded on an X-ray

diffractometer (Instrument Details-Manufacturer: Panalytical Model: Xpert PRO MPD

Anode: Copper K-alpha Wavelength: 1.5405 Angstorm Power: 45KV and 40mA Detector:

Xcelerator with Diffracted Beam) at Tata institute of fundamental Research, Navvy nagar,

Colaba, Mumbai.

The samples were scanned between 2Ѳ=3°and 60° at the scan rate of 4°/min.

Gram staining test

The bacterial smear was prepared by applying a thin smear of bacteria on the glass slide and

left to dry [15]

. The smear of bacteria was fixed to the slide by passing the slide a few times

over a flame. A crystal violet solution was then put on the slide for 1 min and the excess

crystal violet solution on the slide was gently rinsed off with tap water. The gram's iodine

solution was applied to the slide for 1 min and the slide was quickly decolorized with 95%

ethanol. The slide was stained with a counter stain safranin for 1 min and then washed and

dried. Finally, the slide was observed under a light microscope [Coslab (HL-10), Mumbai]

with a magnification of 1000x. The shape and the color of the cell were observed and

photographed using the camera attached to the microscope [Abbot (DEC-200), India].


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Catalase test

To perform this test, a single isolated colony was streaked on a glass slide and one drop of 3

% hydrogen peroxide was added on it. If the effervescence of oxygen indicated the positive

response of the bacteria to catalase test [16]

.

Bile salt tolerance

The ability of the strains to tolerate bile salts was determined using Bile salt tolerance [17]

.

L.actobacillus sporogeness was tested for rapid growth in GYEagar medium with and

without addition of bile salts. GYEagar medium was prepared with different concentrations

of bile salts at 0.1, 0.3, 0.5 and 1.0 % w/v and dispensed in test tube of 10 ml capacity and

sterilized at 121C for 15 min. Two tubes of each concentration were inoculated with 0.1 ml

of Lactobacillus sporogeness culture and incubated at 37C for 48-72 hours. The total viable

counts of Lactobacillus sporogeness were obtained for all concentrations. The results were

expressed as the percentage of growth in presence or absence of bile salts. The bile tolerance

(%) was calculated using the equation.

Quantitative test for lactic acid Production

To an accurately weighed 1 gm of Lactobacillus sporogeness powder, add Exactly 100 ml of

sterile normal saline solution and mix for a 10 min. This makes test dilution of 1:100

dilutions. Transfers 10 ml of test solution in a sterile test tube and allow it to stand in water

bath for 30 min at 75C. Cool immediately to 40 to 45 C. Pipette out Exactly 1ml of solution

in 10 ml sterilized culture media and cooled it at room temperature. Incubate it at 37 C for

48 hours.

After incubation, test solution was titrated with 0.05 N Sodium hydroxide using bromothymol

blue as an indicator. Perform blank in same way by omitting test sample.

E.coli detection and other coli form of bacteria in Lactobacillus sporogeness powder

Weigh 1 g dry Lactobacillus sporogeness powder and mix with 10 ml of sterile water in a 20

ml tube using blender. Add 1 ml of this suspension into a sterile petridish and pour 10 ml of

Equation (1)

Equation (2)


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GYEagar medium, mix well and allow it to set. Then add 10 ml of GYEagar medium to

cover the earlier layer, spread evenly and allow it to set, then incubate at 35-37 °C for 24

hours. Run assay in triplicate (n=3).

Loss on Drying

1 gm of Lactobacillus sporogeness powder was taken and dried in oven for 3 hours.

Percentage loss of weight from previous weight should not more than 5.0%.

Aqueous pH- stability study

The stability of Lactobacillus sporogeness was evaluated in aqueous media of pH range from

1.2 to 8.0 to cover the pH environments encountered by Lactobacillus sporogeness in the

GIT. 1% w/v aqueous suspension of by Lactobacillus sporogeness was prepared in 0.1N HCl

(pH 1.2), 0.01N HCl (pH 2.0), buffers of pH 4.5 acetate, 6.8 and 7.4 phosphate at room

temperature. Suspensions were stirred and analyzed at 0, 1 and 2 hours for microbiological

assay [18]

.

Flow property studies [19]

Density measurement methods

The bulk density was calculated by measuring the mass per unit volume Lactobacillus

sporogeness powder was passed through sieve to break the clumps, if any.Accurately

weighed 50 g of the drug was placed in a 100 ml graduated measuring cylinder. Initial

volume was observed. The bulk density (ρ) was calculated using the following equation.

Where m: mass of the Lactobacillus sporogenes powder

v: volume of the Lactobacillus sporogenes powder

For the tapped density measurement, The cylinder was tapped initially 500 times from a

distance of 14±2 mm. The tapped volume (Va) was measured to the nearest graduated unit.

Again the tapped volume was measured to the nearest graduate unit.

Equation (3)

)

Equation (4)

Equation (5)


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Where,

m: mass of the Lactobacillus sporogene powder.

v: Tapped volume of the Lactobacillus sporogenes.

Hausner Ratio (HR)

Table 1:Scale of flowability Table 1:Scale of flowability

Angle of repose

(degrees) Hausner ratio Type of flow

25–30 1.00-1.11 Excellent

31–35 1.12-1.18 Good

36–40 1.19-1.25 Fair

41–45 1.26-1.34 Passable

46–55 1.35-1.45 Poor

56–65 1.46-1.59 Very poor

>66 >1.60 Very very poor

Compatibility studies: [20]

Excipients are integral components of almost all pharmaceutical dosage forms. The

successful formulation of a stable and effective solid dosage form depends on the careful

selection of the excipients, which are added to facilitate administration, promote the

consistent release and bioavailability of the drug and protect it from degradation.

Compatibility study is the most important part of any pre-formulation testing of proposed

dosage form, and it is necessary that it should be carried out before the development of first

formulation of proposed dosage form with a new drug or new formulation of existing API.

This is required due to the following reasons

Formulation stability studies are time consuming and expensive

Need to minimize the number of model formulations

Provide a rational basis for selecting excipients used in model formulations

Equation (6)

Equation (7)


847


Goal of drug-excipients compatibility study are as follows

1. To find out the excipients that are incompatible with the API

2. To find out that excipients do not have any impact on the stability of the API

3. To find out the excipients that can stabilize the unstable API

4. To assign a relative risk level to each excipients within a functional

5. To design and develop selective and stability-indicating analytical methods to determine

the impurities, wherein the dosage strength difference is very large.

The Lactobacillus sporogeness: Excipients blend were taken in ratio of 1:1 respectively (Fig.

1) and stored at temperature of 40 ± 20 °C and 75 % RH for 30 days. Initial assay of each

blend determined and considered as 100 %. The assay of these blends determined at the end

of study was expressed as percentage of the initial assay. [21]

Figure 1: Schematic representation of compatibility studies

RESULTS AND DISCUSSION

Different selective growth medium

The morphology and characteristics of Lactobacillus sporogeness in different selective

growth medium are presented in Table 2.The properties of the colony were similar in all the

culture media, but colony viable count results were varied in respective media are shown in

Fig. 2.

Table 2:Evaluation of Lactobacillus sporogeness in different growth medium

Morphology and

Characteristics

Culture medium

MRS Agar GYE Agar Nutrient Agar

i) Colony Single Single Single

ii) Shape Circular Circular Circular

iii) Surface appearance Smooth Smooth Smooth

v) Size Big Big Big

vi) Colour White White White

vii) Gram staining +ve +ve +ve

DRUG

1:1 RATIO

EXCIPIENTS

Microbial Assay

No Interaction

iiiiInteractionInt

eraction

Interaction

ALTERNATIVE

EXCIPIENTS

Excipients

Recommended


848


MRS Agar GYE Agar Nutrient Agar

Figure 2: Evaluation of Lactobacillus sporogenes in different growth medium

There were a statistically significant difference in the viable count results in all culture media

but not too much difference in the viable count results among MRS agar and GYE Agar

Medium. Although the viable count of Bacilluscoagulans cultivated in Nutrient medium was

relatively lower as compared with other media as shown in Fig. 3.

Figure 3: Comparative colony viable count of Lactobacillus sporogeness in different

culture medium

Effect of inoculation method

Fig. 4 shows the viable counts of Lactobacillus sporogeness, cultivated by pour plate and

spreading plate method in GYE Agar medium.


849


Pour Plate Technique Spread Plate Technique

Figure 4:Effect of inoculation method on viable count of Lactobacillus sporogeness

The number of viable count of Lactobacillus sporogenes on pouring plate were higher than

that of the spreading method.Comparative results showed that the pour plate method was

more superior than the spreading method (Fig. 5).

Figure 5: Comparative viable count of Lactobacillus sporogenes in pour plate and

spreading plate methods

Microscopic Examination

The spore is seen as small terminal oval or rod shaped retractile bodies at the end of each

vegetative cell (Fig. 6), which complies with Lactobacillus sporogeness specification.

Figure 6: Microscopic Examination of Lactobacillus sporogenes powder


850


Surface Morphology using Scanning electron microscopy (SEM)

Scanning electron microscopy images of Rod shaped Lactobacillus sporogenes probiotic

were shown in Fig.7...

Figure 7: SEM Image of Rod shaped Lactobacillus sporogenes Probiotic

X-ray diffraction study

The XRD study facilitates to realize the crystalline or amorphous nature of core material in

the polymeric matrix. The X-ray diffractograms of Lactobacillus sporogenes (fig. 8),

Lactobacillus sporogenes exhibited multiple characteristic sharp peaks at 2Ѳ varying from

5°to 50°which were due to their crystalline nature.

Figure 8: XRD of L.Sporogenes

Gram Staining Test

The isolated bacteria were observed by light microscope [Coslab (HL-11), India] after gram

staining and the photograph is shown in Fig. 9. It is clear that the bacteria were gram positive,

rod shaped coccobacilli, occurring singly or in chains. The gram staining results indicate that

the isolated bacteria could be identified as Lactobacilli


851


Figure 9: Gram Staining Test of Lactobacillus sporogeness

Catalase Test

No bubble was observed indicating that the isolated bacteria were catalase negative and could

not mediate the decomposition of H2O2 to produce O2. lt is well known that Lactobacillus

acidophilus is catalase negative.

Bile Tolerance Test

Effect of different concentrations of bile salts on the growth of Lactobacillus sporogeness

Mean ± SD, n= 3(Table 3).

Table 3 :Effect of different concentrations of bile salts on the growth of Lactobacillus sporogeness

Bile salt concentration (%) Viable count (CFU/ml) x 107 Bile Tolerance (%)

0.0 153 ±3 100.0

0.1 134 ±2 87.58

0.3 116±2 75.81

0.5 107±3 69.93

1.0 87±2 56.86

Table 4: Physico-chemical characterization study of Lactobacillus sporogenes

Sr.No. Test Specification Result

1

Description Free flowing white to grayish

white powder with

characteristics odour and

slightly sweet in taste

Free flowing grayish white

powder with characteristics

odour and slightly sweet in

taste

2

Identification The spore are seen as small

terminal oval shaped retractile

bodies at the end of each

vegetative cell

The spore are seen as small

terminal oval shaped

retractile bodies at the end of

each vegetative cell

3

Lactic acid

producing capacity

Not less than 10 ml of 0.05 N

sodium hydroxide should be

consumed.

12.6 ml of 0.05 N sodium

hydroxide was consumed.

4 Loss on Drying Not more than 5.0% 3.1%

5 Viable spore count Not less than 10 billion 11 billion spore per gm


852


spore/gm

6

Microbial limit test:-

Test for Presence of

E-coli

Should be absent/10g

absent

Aqueous pH- stability study

For Lactobacillus sporogeness to be beneficial to the host they should colonize in the colon.

Following ingestion, Lactobacillus sporogeness must survive the transit through the gastric

environment and reach the colon in quantities large enough to facilitate colonization.

As seen in Fig. 10 and Table 5, pH of aqueous media had a significant effect on activity of

Lactobacillus sporogenes. Viability of Lactobacillus sporogenes was reduced in acidic as

well as in alkaline pH environment. Lactobacillus sporogenes showed optimum stability at

pH 6.8 condition. Study on the spore forming lactic acid bacillus had observations for acidic

pH environment and found that the spore forming lactic acidbacillus are sensitive to low pH

environment.

Table 5:Aqueous pH- stability study of Lactobacillus sporogenes

Time(hr) %Assay Remaining

pH 1.2 pH 2 pH 4.5 pH 6.8 pH 7.4

0 96.6±0.7 97.8±0.9 98.5±0.6 100±0.4 99.6±0.6

1 82.5±1.2 88.4±0.5 92.5±0.8 99.7±0.7 94.4±0.8

2 76.7±0.7 81.4±0.5 85.8±0.6 98.6±0.8 90.3±0.6

Figure 10: Aqueous pH stability of Lactobacillus sporogenes (Mean±SD, n=3)

Flow property

Flow property from density measurement study, bulk density was found 0.33 and tapped

density 0.370. So, Hausner ratio calculated using equation 7 was found 1.12 shows good flow

property.


853


Compatibility Studies

The blend of Lactobacillus sporogenes and excipients stored at temperature of 40 ± 2 OC and

75 % RH for 30 days. The % recovery of drug after 15 days and 30 days from the

Lactobacillus sporogenes and the excipients blend has been analyzed. It was found that all

results are shown more than 75 % recovery, which shows excipients were compatible with

Lactobacillus sporogenes and they were tabulated in Table 6and shown in Fig.11.

Table 6 :Compatibility study after 15 and 30 days stored at temperature of 40 OC/75% RH

Figure 11: Compatibility study after 15 and 30 days stored at temperature of 40 °C/75%

RH

CONCLUSION

L. sporogenes were found to be sensitive to the conditions encountered in processing of

pharmaceutical and food products. The aqueous pH stability profile showed a rapid

degradation with maximal stability of L. sporogenes at pH 6.8. The spores were found to be

compatible with the excipients evaluated, with noted exception of Sodium alginate, HPMC

K4M, Sodium CMC, Carbopol 934 P. The physicochemical profiling of L. sporogenes

presented in the study provides understanding of the material attributes critical to product

design in terms of selection of formulation ingredients, process conditions and pack

suitability.

Sr.

No Blend(L. sporogenes:Polymer)

% Recovery

15 Days 30 Days

1 Sodium alginate (1%w/v) 81.5 82

2 HPMC K4M 80 79

3 Sodium CMC (5000 cps) 83 84.5

4 Carbopol 934 P 82 80


854


ACKNOWLEDGEMENT

Authors would like to acknowledge Dr.Girish K.Jani Principal, SSR College of Pharmacy,

Silvassa and Mr.Vipul D.Prajapati Associate Professor, SSR College of Pharmacy, Silvassa

for providing support and facilities for this Research studies.

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