INTRODUCTION:

Diarrhoea is one of the major public health problems particularly in developing countries like Bangladesh, India and Pakistan. It is an important cause of a large number of morbidity and mortality especially among infants and children. It has been estimated that 1.3 billion episodes of diarrhoea occurs every year and 3.2 million of children under the age of 5 years die of diarrhoea¹. The magnitude of diarrhoeal disease put a heavy burden on the meager health facilities and resources of a developing country like Bangladesh which is highlighted by the fact that about 30% of paediatric beds are occupied by children with diarrhoea² and 405 cases are treated with unnecessary and inefficient drugs³.

Diarrhoeal disease is very common in developing world, where one out of every 20 children born are destined to die from diarrhoeal dehydration before reaching the age of five. Indeed, it is the major single cause of death among children, and accouts for the death of about five million children a year. In addition, repeated diarrhoea episodes could impair the nutritional status of the affected children who become increasingly susceptible to other acute infections⁴. The great majority of these deaths could be prevented at very low cost⁵.

It is clear that it is both medically sound as well as cost-effective to encourage methods of assessment and treatment of diarrhoeal disease that focus on the recognition and correction of dehydration, rather than on costly interventions.

In diarrhoea, use of ‘Oral Rehydration Salt (ORS) solution’ containing glucose started over more than two decades world wide. The glucose based ORS solution (glucose-ORS) corrects electrolyte imbalance and compensates fluid loss; consequently saves many lives. So the importance of glucose in diarrhoea is admitted indeed. But being costly, addition of glucose rises up the total cost of ORS and makes it hard to purchase. The situation gets worse in a developing country like Bangladesh where the poor constitutes a major fraction of the total population. So researches started and are still continuing to replace glucose in ORS. An ideal substitute of glucose (to be used in oral rehydration salt mixture, in sachets), use of rice of various form has got the preference, but it is under clinical investigations till today and unfortunately no commercial formulation containing pop rice powder has yet been in the market. To prove pop rice powder to be commercially as pharmaceutical dosage form like ORS preparations, studies on their physic-chemical properties and microbiological quality have been conducted here.

In this present study, a new type of rice (pop rice from Gios paddy) was used as a substitute of glucose to formulate ORS solution. The physicochemical and microbiological tests were performed at every 2 hour intervals upto 8 hours in order to conclude whether this solution can be effectively used for the treatment of diarrhoea.

Table-1: Formulation of pop rice based ORS

Components	Amount taken on % basis	gm/500 mL of drinking water
NaCl	6.09	1.75
NaHCO₃	4.35	1.25
KCl	2.61	0.75
Pop rice powder	86.96	25
Total	100	28.75

Figure 1: Photograph of pop rice (Khoi).

MATERIALS AND METHODS:

Materials:

The equipments used in this study were oven (WTB binder-980436, Germany and Memmert, Germany), autoclave (model-Mo-40, Japan), laminar flow cabinet (model ER-17, Japan), incubators (model BE-200), weighing balance (Mettler balance, model-AY 1502, Switzerlamd), pH meter (model-C18, WPA, England), distillaor (type 2004, Germany), electrolyte analyzer (Beckman Coulter TM, USA), centrifuger (Sigma laboratory centrifuger-4K15, USA), microosmometer (Advanced Instruments, INC, USA), microscope (Ortholux Research Microscope, Germany), viscometer (Brook-field Synchro-Lectric viscometer, LVT), bulk density apparatus (model-STAMPFVOLUMETER-STAV-2002). Growth media used here were plate count agar (PCA), potato dextrose agar (PDA), lauryl tryptose broth (LTB), brilliant green bile broth 2%, EC broth, EMB agar, lactose broth, selenite cystine broth and bismuth sulfite agar.

Methods:

Preparation of pop rice powder:

Pop rice is usually produced from paddy, by putting with heated sand. But sand is harmful to human body. To avoid this, three local varieties of paddy (Gios, Kazol and Gura dhan locally called in Laxmipur) were taken, those are most commonly used to produce pop rice in villages. These paddies were put into heated pan without sand for 40 to 50 seconds with continuous stirring. It was separated from husk popped rice that was already detached during heating (figure 1). After separating the husk, the pop rice was crushed by using blender and passed through a sieve (mesh size# 100). Then the powder was used in the newly developed formulation of ORS (Table-1).

Figure 2: Photograph of pop rice based ORS solution.

Manufacturing procedure of pop rice based ORS solution:

Whether the pop rice based ORS is produced in small quantities or in large quantities (in industries) the basic procedure nominees the same. The only difference is the storage facilities for raw materials and packing for finished products (i.e. ORS sachets but two separate chambers, one chamber for salts and another for pop rice powder). Pop rice based ORS was manufactured as per WHO guidelines. All raw materials for ORS production were identified by a Government owned organization or quality control laboratory at first. Raw materials were dried at specific temperature as given bellow:

Pop rice powder max. at 105⁰C

Sodium chloride max. at 130⁰C

Potassium chloride max. at 130⁰C

All of these raw materials may not have the same degree of free-flowing properties. Therefore, they need chopping and grafting. The suitable sizes of perforations on the grafting drum were 3.0 mm and 6.0 mm. Sifting and sieving were carried out to obtain uniform particle size and to screen off foreign particles such as fibre, wood and husk to ensure absence of contaminant most likely to occur in the product. Then the materials were mixed uniformly keeping variables like mixing time and densities of the components same and particle size was kept below 100µ. Pop rice powder and salt mixture were packed in the same sachet but separated from each other. Heat welding method was used for sealing. After proper filling and sealing, the assembly was appropriately labeled and made it ready for use^6-8.

Quality control test of the pop rice based ORS solution

Physical tests:

a) Physical nature: Physical nature and physical appearance of the product was observed after reconstitution in normal water (boiled but normal in temperature).

b) Weight checking: The contents were removed and weight to whether the same was claimed.

c) Sealing: It was done to check whether the sealing was air tight or not.

Figure 3: Total bacterial count of pop rice based ORS solution for every two hour interval in 8 hour duration.

Determination of pH:

After reconstitution in normal boiled water the pH of rice ORS solution was determined potentiometrically using glass electrode and a digital pH meter.

Determination of viscosity:

The viscosity of the prepared ORS solution after reconstitution in normal water was determined by using a viscometer at temperature 24^OC⁹.

Determination of electrolytes (Na⁺, K⁺, Cl^-, HCO₃^-):

The entire content (28.75 g) of the packet was reconstituted in 500 mL of the deionized water and then centrifuged by centrifuge to get clear supernatant solution which was introduced to the chamber of the electrolyte analyzer (Bechman Coutler^TM ,USA) to analyze electrolyltes^6-8.

Determination of osmolarity:

The entire content (28.75 gm) of the packet was reconstituted in 500 mL of the deionized water and then centrifuged by centrifuge to get clear supernatant solution which was introduced to the chamber of The Advanced^TM microosmometer (Advance Instruments, INC, USA) by small tube to determine the osmolality of the solution¹⁰.

Determination of microbiological quality:

1. Preparation of sample for test: 10 gm of sample was added to 90 mL Ringer’s solution. The sample included paddy and pop rice powder and the microbiological quality tests were completed by following standard methods.

2. Determination of standard plate count (SPC): SPC was obtained by standard pour plate method (ICMSF) and for this purpose plate count (PCA) was used as the culture medium. In this counting method, a measured volume of liquid inoculum was placed in a Petri dish and a molten agar medium was added to it at 45-48^OC, which was then mixed to disperse the inoculum throughout the medium. After solidification, it was incubated. In this experiment, firstly a serial dilution was made upto 10^-2 of the original sample using Ringer’s solution as a diluting agent. 1 mL of each dilution was taken on to sterile petridish and molten PCA mediun was poured on to them at 45-48^OC. After solidification, the plates were incubated for 24-48 days at 37^OC. After the recommendation period of time of incubation, colonies were counted from these petridishes containing 30-300 colonies per plate. The actual number of microbial colonies of every sample was calculated per mL by multiplying the number colonies on each plate with their respective dilution factor. It was noted that the replications for each dilution were made to obtain the result as accurate as possible¹¹.

Figure 4: Total fungal count of pop rice based ORS solution for every two hour interval in 8 hour duration.

3. Determination of total coliform: There are two procedure for determination of total coliform-most probable number (MPN) method and membrane filter (MF) method. MPN method¹² was used as it was convenient. The series of tests (presumptive test, confirmed/confirmatory/eijkman test, completed test and biochemical tests for further confirmation) test were performed using various selective and differential media¹³.

4. Total count of fecal coliform: The total colifrom count alone does not always indicate the sample the sample containing fecal materials. But the determination of fecal coliform count definitely indicated whether the sample conytains fecal materials. Although it can be determined preliminary from EMB plate (small, dark centered with greenish metallic sheen 0 during total coliform count), further confirmation was done by MPN method using a selective media at higher temperature that used formarly.In this experiment a loopful inoulum from each of the gas positive lauryl tryptose broth was transferred to the fermentation tubes were incubated at 44.5±0.2^OC for 24±2 hours. The selective medium and the applied temperature only permit the fecal colifrom to grow and form gas in Darham’s tubes during the incubationperoid. After incubation, the gas positive tubes were recorded as positive and the gas negative tubes were obtained by using the standard statistical MPN-table.

Table 2: Physico-chemical and microbiological quality done on pop rice based ORS solution after reconstitution.

Test	Observation	Official specification	Remarks
1. Physical nature after reconstitution
a. Color:	Off-white	NOS	The sample was pop rice ORS solution
b. Form:	Coarse dispersion
c. Odor:	Characteristic
d. Taste:	Salty
2. Viscosity (cps)	200 at 25^OC	NOS	Easily pourable
3. pH	8.3*	7-8.8**	Do
4. Electrolytes in ORS	Mmol/L	Mmol/L
a. Na⁺	90.0*	83.2-94**	Do
b. K⁺	20.2*	18.7-21.5**	Do
c. Cl^-	83.5*	74.4-85.6**	Do
d. HCO₃^-	29.0*	27.7-31.9**	Do
5. Osmolarity (mOsmol/L)	213*	300 or less***	Do
6. Microbiological analysis
a. Standard Plate Count (cfu/mL)	1.8X10²*	10X10^2Ψ	Complied
b. Total Fungal Count (cfu/mL)	1.7X10¹*	10X10^1Ψ	Do
c. Total Coliform Count (MPN/mL)	Not found	Must be nill	Do
d. Total Fecal Coliform Count (MPN/mL)	Not found*	Must be nill	Do
e. Detection of Salmonella	Not found*	Must be nill	Do

*Average of three, **WHO-85, ***WHO-92, Ψ BP 98, NOS = No official specification.

Table 3: Determination of pH of pop rice based ORS solution at every 2 hours interval in 8 hour duration.

time interval (hour)	pH	Official Specification	Remarks
0	8.31*	7-8.8**	pH is slightly changed but within the limit
2	8.45*
4	8.42
6	8.42
8	8.42

* Average of three samples, ** WHO 85.

Table 4: Determination of osmolarity of pop rice based ORS solution for every 2 hour interval in 8 hour duration.

time interval (hour)	Osmolarity (mOsmol/L)	Official Specification	Remarks
0	213*	300 or less**	Osmolarity of the solution was not changed but within the limit
2	213*
4	213*
6	213*
8	213*

* Average of three samples, ** WHO 92.

Table 5: Determination of electrolyte balance of the pop rice based ORS solution at every 2 hours interval in 8 hour duration.

time interval (hour)	Electrolytes in ORS (mmol/L)
	Na⁺	K⁺	Cl^-	HCO₃^-
0	90.1*	21.21*	83.5*	29.1*
2	90.1*	21.2*	83.5*	29.1*
4	89.6*	21.2*	83.6*	28.1*
6	89.6*	21.19*	83.9*	28.8*
8	90.8*	21.17*	84.6*	29.0*
Official Specification	83.2-94**	18.7-21.5**	74.4-85.6**	27.7-31.9**
Remarks	Very small change but complied	Do	Do	Do

* Average of three samples, ** WHO 85.

Table 6: Microbiological quality assessment of pop rice based ORS solution for every 2 hour interval in 8 hour duration.

Test	Time interval (hours)					Official Specification	Remarks
Test	0	2	4	6	8	Official Specification	Remarks
Standard Plate Count (cfu/mL)	2.5X10²*	3.5X10²*	5.X10²*	8.5X10²*	79.33X10²*	Maximum 10X10²**	After 6 hours total bacterial load was very high
Total Fungal Count (cfu/mL)	2.3X10¹*	2.6X10¹*	5.6X10¹*	8.6X10¹*	3.33X10²*	Maximum 1X10²**	After 6 hours total fungal load was very high
Total Coliform Count (MPN/mL)	Not found	Not found	Not found	Not found	Not found	Must be nil	Complied
Total Fecal Coliform Count (MPN/mL)	Not found	Not found	Not found	Not found	Not found	Do	Do
Detection of Salmonella	Not found	Not found	Not found	Not found	Not found	Do	Do

Average of three samples, ** BP 98.

5. Qualitative detection of Salmonella spp: Pre-enrichment of the sample in a nutritious nonselective broth, subsequent enrichment in a selective broth, isolation of pure culture of Salmonella by streaking on to a selective agar plate, biochemical characterization and definitive serological confirmation of biochemical screened isolates¹³.Lactose broth was used as the pre-enrichment media recommended by American Public Health Association (APHA), International Commission on Microbiological Specifications for foods (ICMSF) etc. In this step 25 gm of sample was added to 175 mL of sterile lactosebroth, than it was incubated at 37^OC for 24 hours. Selenite cystine broth was used as selective enrichment medium which was most widely used. In this step one mililitre of pre-enrichment culture was transferred to 10 mL of sterile selenite cystine broth medium, then incubated at 37^OC for 24 hours. In enrichment step, a selective medium and environmental conditions were used which encouraged the growth of required organism but unfavourable for the growth of undesired organism¹².

6. Enumeration of total fungal count: Potato dextrose agar (PDA) plates were used for enumeration of total gungi. Incubated plates in duplicate were incubated at room temperature for 78 hours. After incubation, total fungal load was determined.

The effect of storage condition on the physicochemical properties of pop rice based ORS solution:

To study the effect of storage on physicochemical properties of components present in ORS appearance, pH, bulk density, compositional analysis and microbial analysis were done on the finished product for three months.

RESULTS:

Physicochemical properties and microbiological quality assessment of pop rice based ORS solution:

From the results given in table 2, it was observed that the sample solution was off-white coarse dispersion and possess characteristic odor and salty taste. The viscosity of the sample solution was observed 200 cps at 25^OC which was found easily pourable. Electrolytes concentration tests was carried out and observed value for Na⁺, K⁺, Cl^- and HCO₃^- were 90 mmol/L, 20.2 mmol/L, 83.5 mmol/L and 29.0 mmol/L respectively and the values were acceptable limit in comparison to standard.Osmolarity of the sample was obtained 213 mOsmol/L which was within the range. Furthermore pH of the sample was tested and the value obtained 8.3 which was within the range standard.

For microbiological quality assessment, the reconstituted solution of the pop rice based ORS sample was analyzed and it was found that the Standard Plate Count (1.8X10² cfu/mL) and total fungal count (1.7X10¹ cfu/mL) were within the acceptable limit in comparison with the standard (table 2) but the total coliform, fecal coliform and Salmonella were not recorded at all (table 2).

Physicochemical properties of pop rice based ORS solution during 8 hours period after reconstitution: Physicochemical tests of pop rice based ORS solution was carried out for 8 hours and at every 2 hours and the results were recorded in table 3, 4 and 5. During that period pH was slightly changed but within the acceptable limit and the pH value for 0, 2, 4, 6 and 8 hour were observed as 8.31, 8.45, 8.42, 8.42 and 8.42 respectively.

Electrolytes concentration was tested for the pop rice based ORS solution for every two hour interval and the Na⁺ values for 0, 2, 4, 6 and 8 hours were observed

90.1mmol/L, 90.1 mmol/L, 89.6 mmol/L, 89.6 mmol/L and 90.8 mmol/L respectively but change of the concentration of Na+ concentration was found as negligible. The concentration of K⁺ value for 0, 2, 4, 6 and 8 hour were observed as 21.21 mmol/L, 21.2 mmol/L, 21.2 mmol/L, 21.19 mmol/L and 21.17 mmol/L respectively. The concentration of Cl^- was examined during 8 hour period and the value for every 0, 2, 4, 6 and 8 hour were observed as 83.5 mmol/L, 83.5 mmol/L, 83.6 mmol/L, 83.6 mmol/L and 84.6 mmol/L respectively. The HCO₃^- concentration was also studied during 8 hour period and the value for every 0, 2, 4, 6 and 8 hour were observed as 29.1 mmol/L, 29.1 mmol/L, 28.1 mmol/L, 28.8 mmol/L and 29.0 mmol/L respectively but the difference was found as negligible (table 4).

Furthermore, osmolarity of the pop rice based ORS solution was also tested for 8 hour period and the value at every 0, 2, 4, 6 and 8 hour were onserved as 213 mOsmol/L and the value was remained same.

Microbiolical quality of pop rice based ORS solution during 8 hours period after reconstitution: Microbiologial studies of the reconstituted solution of pop rice based ORS was carried out for every 2 hour interval upto 8 hours time period. Standard Plate Count for 0, 2, 4, 6 and 8 hours were observed as 2.5X10² cfu/mL, 3.5X10² cfu/mL, 5X10² cfu/mL, 8.5X10² cfu/mL and 7.933X10³ cfu/mL respectively. So it is clear that up to 6 hours Standard Plate Count was within acceptable limit (table 6 and figure 3).

Total fungal count was tested and the count for 0, 2, 4, 6 and 8 hour were observed 2.3X10¹ cfu/mL, 3.6X10¹ cfu/mL, 5.6X10¹ cfu/mL, 8.6X10¹ cfu/mL and 3.33X10² cfu/mL. The results showed that upto 6 hours total fungal count was within acceptable limit (table 6 and figure 4). Furthermore, total coliform count (MPN/mL), total fecal coliform count and Salmonella were found absent during 8 hour period (table 6).

DISCUSSION:

Pop rice based ORS after reconstitution forms a dispersion and pH of the sol was found to be slightly basic (pH 8.31) and viscosity of the solution was 200 cps at 25^OC. In this value the preparation could be easily swallowed by the children like an usual drink. The children community might accept it willingly, if appropriately colored and flavored. It was suspected that after reconstitution, the elemental composition may undergo changes in its physicochemical parameters. So, quantitative and qualitative tests were done on pop rice based ORS water dispersion. Results of the tests complied with the official values. The observed values found for the elements, that is Na⁺, K⁺, Cl^- and HCO₃^- present in the samples were 90.1, 20.2, 83.5 and 29.0 mmol/L respectively which are within the official recognized values (in mmol/L value) for Na⁺, K⁺, Cl^- and HCO₃^- respectively. Osmolarity of the pop rice ORS solution was determined by osmometer. The value was 213 mOsmol/L. The official value is 300 mOsmol/L, which was within the limit.

Microbiological tests on total counts for bacteria, fungal and pathogenic bacteria (coliform, fecal coliform and Salmonella) were done at different time intervals to show the maximum permissible time limit to use the formulation. Results showed numerous bacterial and fungal growth take place in the sample solution after 6 hours at room temoerature and indeed to be discarded after the time period and further to be observed.

ACKNOWLEDGEMENT:

Authors are thankful to Bangladesh Council for Scientific and Industrial Research (BCSIR) and International Centre for Diarrhoeal Disease Research Bangladesh (ICDDRB) for providing materials and laboratory facilities.

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Received on 27.04.2009 Modified on 22.06.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 572-577