evaluation of microbiological and physicochemical profile of

Transcription

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Himanshu et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 4, Issue 03, 839-855.
Research Article
ISSN 2278 – 4357
EVALUATION OF MICROBIOLOGICAL AND PHYSICOCHEMICAL
PROFILE OF PROBIOTIC LACTOBACILLUS SPOROGENES AS A
BIOTHERAPEUTICS AGENT
Himanshu K. Solanki1* and Dushyant A. Shah2
1
Department of Pharmaceutics, S.S.R. College of Pharmacy, Sayli-Silvassa Road, Sayli,
Silvassa, U.T. of D.N.H.-396230, India.
1
Ph.D Scholar, Hemchandracharya North Gujarat University, Patan-384265, Gujarat, India.
2
APMC College of Pharmaceutical Education and Research, Motipura, Himmatnagar383001, Dist: Sabarkantha.India.
Article Received on
20 Dec 2014,
Revised on 13 Jan 2015,
Accepted on 07 Feb 2015
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,
*Correspondence for
processing,
storage
and
in
vivo
environment.
Lactobacillus
Author
Himanshu K. Solanki
sporogenes a revolutionary new friendly bacteria naturally occurring in
Department of
the intestine. It is a spore-forming bacterium that makes it the choice
Pharmaceutics, S.S.R.
of probiotic with
College of Pharmacy,
Lactobacillus sporogenes spores were studied for microbiological
Sayli-Silvassa Road,
enormous
clinical
applications.
Methods:
studies, organoleptic properties, Microscopic examination, Scanning
Sayli, Silvassa, U.T. of
D.N.H.-396230, India.
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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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, sporelike 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 selfaffirmed 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 heattreatments 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,
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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 37C 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 doublesided 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 121C for 15 min. Two tubes of each concentration were inoculated with 0.1 ml
of Lactobacillus sporogeness culture and incubated at 37C 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.
Equation (1)
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 75C. 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.
Equation (2)
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
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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.
Equation (3)
)
Equation (4)
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 (5)
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Equation (6)
Where,
m: mass of the Lactobacillus sporogene powder.
v: Tapped volume of the Lactobacillus sporogenes.
Hausner Ratio (HR)
Equation (7)
Table 1:Scale of flowability Table 1:Scale of flowability
Angle of repose
Hausner ratio
Type of flow
(degrees)
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
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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]
DRUG
No Interaction
1:1 RATIO
Microbial Assay
EXCIPIENTS
iiiiInteractionInt
Excipients
eraction
Recommended
Interaction
ALTERNATIVE
EXCIPIENTS
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
Culture medium
Characteristics
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
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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.
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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
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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
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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.
1
2
3
4
5
Test
Description
Specification
Free flowing white to grayish
white
powder
with
characteristics
odour
and
slightly sweet in taste
Identification
The spore are seen as small
terminal oval shaped retractile
bodies at the end of each
vegetative cell
Lactic
acid Not less than 10 ml of 0.05 N
producing capacity
sodium hydroxide should be
consumed.
Loss on Drying
Not more than 5.0%
Viable spore count
Not less than 10 billion
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Result
Free flowing grayish white
powder with characteristics
odour and slightly sweet in
taste
The spore are seen as small
terminal
oval
shaped
retractile bodies at the end of
each vegetative cell
12.6 ml of 0.05 N sodium
hydroxide was consumed.
3.1%
11 billion spore per gm
851
Himanshu et al.
spore/gm
6
Microbial limit test:Test for Presence of Should be absent/10g
E-coli
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
%Assay Remaining
Time(hr)
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.
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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
Sr.
No
1
2
3
4
Blend(L. sporogenes:Polymer)
Sodium alginate (1%w/v)
HPMC K4M
Sodium CMC (5000 cps)
Carbopol 934 P
% Recovery
15 Days
30 Days
81.5
82
80
79
83
84.5
82
80
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.
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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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