Research articleResearch articlejobb.co.in/docs/vol2_issue2/all.pdf · The plant growth promotion activity of fermented leaf juice of Moringa oleifera (PKM-1) was determined by in-vitro

Journal of Biotechnology and Biosafety

Volume 2, Issue 2, March-April 2014, 61-67 ISSN 2322-0406


www.jobb.co.in International, Peer reviewed, Open access, Bimonthly Online Journal

Research articleResearch articleResearch articleResearch article

MORINGA OLEIFERA (PKM-1) FERMENTED LEAF JUICE- PART OF ORGANIC INTEGRATED NUTRITIONAL MANAGEMENT IN ORGANIC CULTIVATION OF

BRASSICA OLERACEA (L.) _________________________________________________

R. Rajamani1*, Rudresh Kumar Singh2, Lakshmi B3 _________________________________________________ 1Co-principal investigator, Sri Sri Institute of Advanced Research (A Research Division of Ved Vignan Maha Vidya Peeth- VVMVP) 21st Km, Kanakapura Road, Udayapura, Bangalore- 560082

2Research scholar, Sri Sri Institute of Advanced Research (A Research Division of Ved Vignan Maha Vidya Peeth- VVMVP) 21st Km, Kanakapura Road , Udayapura, Bangalore- 560082 3Research associate, Sri Sri Institute of Advanced Research (A Research Division of Ved Vignan Maha Vidya Peeth- VVMVP) 21st Km, Kanakapura Road, Udayapura, Bangalore- 560082 *Corresponding author email: [email protected]

ABSTRACT The plant growth promotion activity of fermented leaf juice of Moringa oleifera (PKM-1) was determined by in-vitro pot culture method. Brassica oleracea (L.) (Knol-khol) plant treated with consortium and Jeevamrit were supplemented with Drumstick fermented leaf juice showed significant growth promotion than un-supplemented plant. Three experimental plants were used; plant treated with 15 ml, 30 ml and 45 ml/plant during vegetative growth period (thirty five days). The biomass parameters such as fresh-shoot weight (73.31 gm), fresh-root weight (24.4 gm), root-length (27.1 cm), dry-shoot weight (1.3 gm), dry-root weight (0.8 gm), total fresh- weight (97.71 gm ) and total dry weight (2.1 gm) were higher in D-3 (45 ml/plant) than control ( un-supplemented). The study revealed that the root application of combination of Jeevamrit, consortium and DFLJ on Knol-khol plant was observed suitable for plant growth promotion. In this paper effective utilization of Drumstick leaf on Knol-khol plant has been described.

Keywords: Moringa oleifera, Jeevamrit solution, Drumstick fermented leaf juice (DFLJ), Brassica oleracea, Micronutrient, green manure, organic cultivation, knol-khol. ______________________________________________________________________________________________________





INTRODUCTION Moringa is a tropical tree with multiple uses and which is resistant to drought. Among the 13 species known, Moringa oleifera is particularly easy to reproduce and its growth is very fast. The numerous economic uses of Moringa oleifera with its easy propagation have raised growing international interest. This tree which originated from India, found in most tropical countries- Africa, Asia, America and Madagascar have been less exploited so far. Though India stands second in vegetables and fruit production hardly two percent of the produce is processed and 30-40% is wasted due to lack of processing and preservation infrastructure (Adeyeye 2002). The Moringa leaves possess remarkable nutritional and medicinal qualities. The leaves contain high amount of Vitamin C, Calcium, Potassium, Proteins and rich essential amino acids such as Arginine and Histidine (Singh, S et al., 2012, Mishra, S P 2011, Duke, J A 1987, Manzoor, M, 2007, Mahatab, S N 1987). India’s ancient tradition of Ayurveda says the leaves of Moringa tree prevent 300 diseases. Scientific research has proven that these humble leaves are in fact a power house to nutritional value. The micronutrient content is even more in dried leaves; 10 times the vitamin A of carrots, 17 times the calcium of milk, 15 times the potassium of bananas, 25 times the iron of spinach and 9 times the protein of yogurt (Moringa News- web news). Moringa can be grown intensively which yields up to 650 metric tons of green matter per hectare. This compares very well to other green manure crop such as Lab-lab beans which yields up to 110 metric tons per hectare. The researchers at Proyecto BIOMASA Agricultural research program located in Nicaragua have studied for over six years that Moringa fresh leaf juice and fresh leaves can be used for sound agricultural practice like foliar spray to increase plant growth and as a green manure to improve soil fertility. The juice from fresh Moringa leaves can be used to produce an effective plant growth hormone (Zeatin- cytokinin group) and increase yield by 25-30% for nearly all crops like onion, bell paper, soya, maize, sorghum, coffee, tea and chilli (Martin L. Price, 2007). Hence the present study was undertaken specially to investigate the role of drumstick fermented leaf juice as an organic nutritional source and growth promoting agent on Brassica oleracea.

MATERIALS & METHODS The leaf of Moringa oleifera PKM-1 was collected from two years old tree at Sri Sri Ayurveda Panchkarma Division (SSAPD) organic garden, Art of Living International Centre, Bangalore, to prepare drumstick fermented leaf juice. Preparation of Drumstick Fermented Leaf Juice Three kg of fresh leaves of Moringa oleifera PKM-1 variety were weighed out and immersed into three litre of tap water containing three grams of salt & three grams of tamarind (Table-1) (Vijayan Pillai, 2012). The whole mixture was kept in 30 litres capacity plastic container and left for fifteen days for aerobic fermentation. After fifteen days, the juice from fermented leaf was filtered off and stored in a clean conical flask for experimental use. Preparation of Nursery Bed for Knol-khol Plant For this study, Brassica oleracea (KNOL-KHOL,``INDAM EARLY WHITE’’) seeds were obtained from Indo-American hybrid Seeds(India) Pvt.Ltd. Fifty seeds were surface sterilized with 5% sodium hypo chloride for 5 minutes and rinsed thoroughly in sterile distilled water for three times. Seeds were sown on a nursery bed containing 1:1:1 ratio of coco peat, red soil and cow dung. After fifteen days the seedlings were transplanted into pot soil mixture. Preparation of Pot Soil Mixture To evaluate the DFLJ effect, a specially designed pot soil mixture was prepared. Two parts of coco peat, one part of red soil and one part of cow dung (2:1:1) were mixed thoroughly and 1 kg of pot soil mixture was filled in each pot. Experimental Design The fifteen days old seedlings of Knol-Khol were transplanted to the pot containing soil mixture along with different quantity of DFLJ (Table-1). Each treatment had ten plants. The first set of 10 pots of Knol-Khol plants were treated with 5mL of DFLJ /plant, second set 10mL/plant and third set 15mL/plant of DFLJ at the time of transplantation. The fourth set of 10 pots were not treated with drumstick fermented leaf juice and treated as control. The same treatment was repeated again on 15th day and 30th day of Knol-khol plant along with 2.5 mL Jeevamrit solution (Table-2) and 750 mg of bacterial and fungal consortium (Product obtained from Indian Institute of Horticulture Research Soil Science, Bangalore).The control was only treated with Jeevamrit solution and consortium. The water requirement of pot culture plants were managed as and when during the experiments.





Table-1: Composition of Drumstick Fermented Leaf Juice (DFLJ). Sl. No Composition

1 3 kg of fresh drumstick leaf 2 3 grams of tamarind 3 3 grams of sea salt 4 3 Liters of tap water

Table-2: Composition of Jeevamrit solution

S. No Composition 1 10 kg of fresh cow dung 2 10 liters of cow urine 3 2 kg of Jaggery 4 2 kg of Red gram flour powder 5 200 grams of pot soil mixture

Analysis of Biomass For biomass analysis, thirty five days old vegetative stage of Knol-Khol plants was taken out from pot carefully and washed with tap water to remove the soil. For fresh and dry biomass analysis, the shoot and root were separated and placed in a paper envelop after weighing the fresh individual plants. The shoot and root were placed in oven at 57oC for three days to remove complete inbound moisture (Gamalero, 2004). The dry weight of shoot and root of individual plants were recorded. The other parameters such as root length and number of leaves were also recorded to study the effect of DFLJ.

RESULTS According to data, the vegetative biomass of knol-khol (Table 3) increased significantly due to different concentration of DFLJ. The highest total fresh weight 97.71 gm. was obtained from the plant treated with 45mL of DFLJ (D3), which was significantly higher over all other treatments. The second highest biomass yield 11.63 gm was obtained from the plant treated with 30mL of DFLJ (D2). The biomass of individual plants shoot-fresh and dry-weight, root-length, root-fresh and dry- weight showed more than control (Table 4). The number of leaves per plant did not show any significant variation.

Table-3: Effect of different dose of D F L J on Knol-khol Shoot Biomass





Figure-1: Effect of DFLJ on shoot biomass of Knol-khol

Table-4: Effect of different dose of D F L J on Knol-khol Root Biomass

Figure-2: Effect of DFLJ on root and total biomass of Knol-khol





Figure-3: Effect of Drumstick Fermented Leaf Juice (DFLJ) on Biomass of Knol-khol plant. A- Control, B- 15 ml/ plant, C- 30 ml/ plant, D-45 ml/ plant.

DISCUSSION Based on the result, it was showed a synergistic effect between apply of Jeevamrit, consortium and DFLJ on biomass content. Many studies showed that application of microbial consortium significantly increased biomass of the plant due to vigorous uptake of minerals from the soil. Increased individual plant biomass in treatment (D-3) might be due to the higher dose of available nutrients of DFLJ. Increased nutrient uptake by plants treated with combination of all three such as Jeevamrit, consortium and DFLJ has been attributed to enhancement of increased root volume and surface area. The composition of DFLJ was

developed and applied on tomato, papaya at AL.Khaly Farm in UAE to withstand high temperature, a better yield was observed.(Vijayan Pillai, 2012, www.nonitrees.com) Combined inoculation of multiple traits of Azospirilum, Bacillus, Pseudomonas, Enterobacter and Azotobacter have also been reported in various crops like tomato, potato, rice, sugar beet and barley (Gamalero, E. et al., 2004, Kundu, B.S. and A.C. Gaur, 1980, Tiwari, V.N et al 1989). The presence of freely available minerals, amino acids in DFLJ might be mobilized at high rate to the plants root with microbial activity. Further research need to be done on mechanism by which DFLJ increased biomass of Knol-khol and other crops in order to find whether other mechanisms are also involved.





CONCLUSION

The present study recommends the prospects of more aggressive introduction and utilization of drumstick fermented leaf juice (DFLJ) by the Agriculture sector. It also implies that it may be worthwhile for small margin farmers to take up the production of drumstick fermented leaf juice for healthy cultivation and incorporated into the Organic Integrated Nutritional Management (OINM) programme in India and other countries. This could go a long way towards not only alleviating plant macro and micro-nutrient deficiencies but also towards the enrichment of soil for several crops. By introducing DFLJ as an organic nutritional source for different crops, surely it sustains the agriculture in future because of proper and regular use of DFLJ by the farmers may stop the use of inorganic fertilizer and reduce the input cost. It is concluded from the present study that the fifteen days old DFLJ, consortium and Jeevamrit mixture showed better activity by enhancing the growth of knol-khol plant. These mixtures can be used as a organic nutritive solution for growth promotion of all crops in the future. Acknowledgement We are grateful to Mr. Prasanth S Nair, Director, VVMVP Trust, Art of Living International Center, Bangalore, India. We also thank Vinoda Kochupillai, Chairperson, SSIAR and Mr. Ghanshyam Srivastava, HOD, SSIAR for providing facilities and showing keen interest for completion of the work. REFERENCES Adeyeye EI (2002). Determination of the chemical composition of the nutritionally valuable parts of the male & female common west African fresh water crab Sudananutes africanus africanus. Int. J. Food Sci. Nutr., 53(3): 189-196. Cakmakci, R., F. Kantar and O.F. Algur, (1999). Sugar beet and barley yields in relation to Bacillus polymyxa and Bacillus megaterium var. Phosphaticum inoculation. J. Plant Nutr. Soil Sc., 162: 437-442. Duke, J.A (1987). Moringaceae: Horse radish- tree, benzolive tree, drumstick tree, sohnja, Moringa, Murunga kai, malunggay, P. 19-28. In: M. Benge (ed) Moringa: a multipurpose vegetable and tree that purifies water. Sci &

Technol. For. / Environ & Natural Resources Forestation Tech. Ser. 27. USAID, Washington, D.C. Gamalero, E., M.G. Martinnoti, A. Trotta, P. Lemanceau and G. Berta, (2004). Morphogenetic modification induced by Pseudomonas fluorescence A6RI and Glomus mosseae BEG12 in the root system of tomato differ according to plant growth conditions. New Phytol., 155:293-300. Kundu, B.S. and A.C. Gaur, (1980). Effect of phosphobacteria on the yield and phosphate uptake of potato crop. Curr. Sci., 49: 159. Mahatab, S.N., Ali, A and Asaduzz aman, A.H.M (1987) Nutritional potential of sajna leaves in goats. Live stock Advisor., 12(12): 9-12. Manzoor, M; Anwar, F; Iqba, T.I and Bhnager, M.I.(2007) Physiochemical characterization of Moringa concanensis seed and seed oil. J. Am. Oi1 Chem. Soc., 84: 413-419. Martin L. Price (2007)The Moringa Tree- ECHO Technical Note 17391, Dwrance Road, North Fort Myers, FL, 33917, USA. Revised 2007 Mishra, S.P., Singh. P and Singh, S.(2011) Nutritional and medicinal values of Moringa oleifera leaves. Potential and Prospects Forestry Bulletin., 11(1): 46-58. Singh, S., Mishra, S.P., Singh, P., Prasad, R.S and Das, R. (2012) Potential and prospects of Moringa oleifera Lam. (Sahjan) Institute of forest productivity., IFP/2012/01. Talukder M R, Banu M B, Hoque A K M S, Houque M A. (2013) Response of knol-khol to different levels of nutrients. Eco-friendly Agril. J., 6(02): 29 Tiwari V.N., L.k. Lehri, A.N. Pathak, 1989. Effect of inoculation crops with phosphomicrobes. Exp. Agr., 25: 47-50. Vijayan Pillai. (2012) Where nature and environment are in a perfect harmony. Gulf Agriculture Trade Magazine- ISSN 1751-8407., 50-52. (www.nonitrees.com) Web site - www.moringanews.org/biblio_en.html.





Citation of this article: R. Rajamani,Rudresh Kumar Singh, Lakshmi(2014) B.MORINGA OLEIFERA (PKM-1) FERMENTED LEAF JUICE- PART OF ORGANIC INTEGRATED

NUTRITIONAL MANAGEMENT IN ORGANIC CULTIVATION OF BRASSICA OLERACEA (L.) JOBB.2(2):61-67.

Source of Support: Nil Conflict of Interest: None Declare


Volume 2, Issue 2, March-April 2014, 68-79

ISSN 2322-0406 Journal of Biotechnology and Biosafety


Research article

ISOLATION AND IDENTIFICATION OF MICROFLORA IN TOBAC CO AND ITS ASSOCIATED FORMS FROM REGIONS OF HOSUR, BANGALORE.

_____________________________________ Dona Saha1, Anwesha Purkayastha2, Prajesh P3* ________________________________________________ 1Microbiologist & Assistant QC incharge at SBPL (Britannia), Bangalore. 2Trainee at Indian Institute of Chemical Biology (IICB), Kolkata. 3Associate professor,Department of Microbiology, The Oxford College of Science,Bangalore.

*corresponding author email id: [email protected]

ABSTRACT:

Tobacco smoking is the practice where tobacco is burnt and the resulting smoke is inhaled which causes various types of diseases and disorders. Disorders are prevalently known in case of practicing tobacco. There have not been found much awareness about microbial diseases from tobacco practice. In this experiment tobacco with its various forms were isolated from region of Hosur, Bangalore which was then screened for presence of bacteria and fungi. This was carried out on the basis of staining and basic biochemical tests. There has been found presence of Klebsiella oxytoca (Gram negative bacteria) and Bacillus (Gram positive bacteria) species and amongst fungal species, occurrence of Aspergillus sp. was established. Microbial presence in tobacco necessarily does not cause diseases but are considered opportunists which may cause disease in immuno compromised individuals.

Keywords: Tobacco, Aspergillus, Klebsiella, Actinomycetes,

___________________________________________________________________________________________________

INTRODUCTION:

Tobacco smoking is the practice where tobacco is burnt and the resulting smoke (consisting of particle and gaseous phase) is inhaled. The practice may have begun early as 5000-3000 BC. Tobacco was introduces to Eurasia in the late 16th century where it followed common trade routes. The practice encountered criticism from its first import into western world onwards, but embedded itself in certain strata

of a number of societies before becoming widespread upon the introduction of automated cigarette rolling apparatus. Smoking is the most common method of consuming tobacco, and tobacco is the most common substrate smoked. The agricultural product is often mixed with additives and then pyrolyzed. The resulting smoke is then inhaled and the active substances absorbed through the alveoli in the lungs. The active substance triggers chemical reactions in nerve





ending, which heighten heart rate, alertness, and reaction time. (Patterson F et al., 2004).

Gram-negative bacteria, mesophilic fungi, thermo tolerant fungi and thermophilic actinomycetes, but not Aspergillus glaucus fungi, were found in higher concentrations in the cigar factory than in the cigarette factories (Exposure to Microbes, Endotoxins and Total Dust in Cigarette and Cigar Manufacturing: an Evaluation of Health Hazards MARJUT REIMAN and JUKKA UITTIJ*).Due to the quality of the raw material used in the tobacco industry, air in the production area of factories is humidified either by humidifiers connected to the ventilation system or by separate humidifiers. These devices can cause microbiological problems if incorrectly maintained. (Rylander and Haglind, 1984).

According to The World Health Organization, there were approximately 1.3 billion smokers worldwide in 2003, and that number is expected to increase to 1.7 billion by 2020.1 It is estimated that about 1 billion people will die from smoking in the 21stcentury. (Michael Rabinoff et al., 2007).

Bacteria found in tobacco products are associated with saprophytic bacteria which may cause opportunistic infection. An opportunistic infection is an infection caused by pathogens, particularly opportunistic pathogens those that take advantage of certain situations—such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system. A compromised immune system, however, presents an "opportunity" for the pathogen to infect that includes Enterococcus, Bacillus sp., Klebsiella, Staphylococci sp., Pseudomonas. (Eng et al., 1991).

Other than microbial infections tobacco practice can also lead to systemic disorders which can lead to many acute and chronic respiratory and pulmonary disorders (Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation). (John L. Pauly and Geraldine Paszkiewicz 2011).

Tobacco products are found with bacterial endotoxin content. Inhalation of bacterial LPS can lead to septic shock in human and endotoxins also produce chronic bronchitis. LAL (Limulus AmoebocyteLysate) assay is used to detect the presence of endotoxin on tobacco products. (Jeffrey D. Hasday et al., 1999).

This experiment was carried out to isolate and identify the microflora present in tobacco and tobacco associated forms from regions of Hosur, Bangalore. Various forms of available tobacco were used and processed initially in physiological saline which after an optimum incubation temperature period was tested for presence of both Gram positive and Gram negative microorganisms with the help of selective media and a range of biochemical tests.

MATERIALS AND METHOD:

Primary screening of Bacteria. (R.P Straka and J.L Stokes, 1957)

The samples obtained were first inoculated in peptone water for 8 hours for enrichment of the culture.

Procedure:

• Required quantity of Nutrient agar is prepared weighing the components as per the composition given and pH is adjusted to 7.1.

• The medium is sterilized using an autoclave in a conical flask for 1210C for 15 minutes at 15lb pressure.

• The media is cooled slightly and poured into sterile petriplates under aseptic condition.

• The media is allowed to solidify.

Secondary Screening and biochemical analysis:

The microorganisms isolated checked for colony morphology and were screened for the biochemical analysis.





Gram’s Staining:(Harley Prescott, 2002, page no. 46) Procedure

• A thin smear of bacterial culture is made on clean grease glass slide.

• The smear is dried and heat fixed. • The smear is flooded with crystal violet for 1

minute. • The slide is washed under running tap water until

all the stain runs off from the slide. • The smear is then covered with Gram’s iodine

solution for 1 minute. • The Gram’s stain is washed off with 70% ethyl

alcohol for 15-20 seconds. • The slide is washed and counter stained with

saffranin for 1 minute. • The slide is then observed under oil immersion

objective. Catalase Test: (Harley Prescott, 2002, p-170) Procedure:

• Small amount of bacterial culture was introduced into 1mL of hydrogen peroxide solution with help of grass rod and observed for release of bubbles.

Oxidase Test :( Harley Prescott, 2002, p-180)

Procedure:

• 2-3 drops of oxidase reagent is added on to a strip of filter paper to moisten it.

• A loop full of test organism is streaked in a zigzag manner on this filter paper.

• A positive reaction is indicated by dark purple colour that developed in 10-15 seconds.

Motility test : (Harley Prescott, 2002, page no.149)

• Motility test was performed for the identification of motility of bacteria using SIM Agar in stab cultures.

BIOCHEMICAL TEST FOR THE GRAM POSITIVE MICROBE: Based on the result the Gram positive microbes were subjected to starch and gelatin test.

Starch Test:(Harley Prescott, 2002, p-140) Procedure:

• Starch agar medium are prepared using the composition given and dispensed into sterile Petri plates.

• Using sterile techniques, a single streak of the test culture is made in the centre of the plate and appropriately labeled.

• The plates are incubated at 370C for 24 hours. A control plate without innoculum is also placed with the lot.

• After proper growth, the plates are flooded with iodine solution and left to stand for 30 seconds. Excess stain is removed and the results are recorded.

GelatinTest:(Harley Prescott, 2002, p-166) Procedure:

• Sterile nutrient gelatin tubes were inoculated with test organisms.

• An uninoculated tube is used as control. • Tubes were incubated at 370C for 24 hours. • After incubation, the tubes are tested for

liquification by placing them in ice bath for 5 minutes and observed for the results.

BIOCHEMICAL TEST FOR MAINLY GRAM NEGATIVE ORGANISMS.

Procedure:(Harley Prescott, 2002, p-80)

• Required quantity of Macconkey agar is prepared weighing the components as per the composition given and pH is adjusted to 7.1

• The medium is sterilized using an autoclave in a conical flask for 1210C for 15 minutes at 15lb pressure.

• The media is cooled slightly and poured into sterile petriplates under aseptic condition.

• The media is allowed to solidify.





Indole Test: (Harley Prescott, 2002, p-155) Procedure:

• The broth is prepared according to the given composition, dispensed in the test tubes and sterilized.

• These tubes were inoculated with test organisms. • The inoculated tubes were incubated at 370 C for

24 hours. • After incubation 1mL of Kovac’s reagent was

added to all the test tubes and the mixture was allowed to stand for 5 minutes and was observed for cherry red ring at the top of the broth.

Methyl Red Test: (Harley Prescott, 2002, p-157) Procedure:

• MR-VP broth is prepared according to the

given composition. The broth is then dispensed into sterile test tubes and sterilized.

• The test organisms are inoculated into different sterilized tubes containing the broth. One tube acts as control without inoculation.

• The tubes are inoculated at 370C for 24 hours. • About 1mL of methyl red is added to the test

culture tubes and observed for the appearance of red or yellow colour.

Vogus Proskaur Test: (Harley Prescott, 2002, p-157) Procedure:

• MR-VP broth is prepared according to the given composition.

• The broth is then dispensed into sterile test tubes and sterilized.

• The test organisms are inoculated into different sterilized tubes containing the broth. One tube acts as control without inoculation.

• The tubes are inoculated at 370C for 24 hours. • After incubation, 6 drops of Barritt’s reagent 1

and 3 drops Barritt’s reagent 2 were added to each of the tubes. The tubes are shaken gently and allowed to stand for 15-30 minutes.

• The change in colour is observed.

Citrate Test: (Harley Prescott, 2002, p-158) Procedure:

• Citrate Agar is prepared according to the given composition, dispensed into test tubes and sterilized and made into slants.

• Test tubes are inoculated with given cultures and were incubated at 370C for 24 hours.

• Tubes are observed for colour change. Endospore Staining for Gram Positive bacteria:(Harley Prescott, 2002, p-58-59) Procedure

• A smear of the organism was prepared on a clean glass slides.

• The smear was air dried and heat fixed. • The smear was flooded with malachite green and

steamed for 5 minutes on a hot water bath without allowing it to dry. A small piece of filter is placed on the smear to prevent rapid evaporation of the stain.

• The slide is cooled; filter paper was removed and rinsed with tap water.

• The smear is counter stained with saffranin for 30 seconds.

• The slide is rinsed with water, dried and examined under oil-immersion objective.

• All the cultures were tested for different carbohydrate fermentation.

Carbohydrate fermentation test: (Harley Prescott, 2002, p-129) Procedure:

• Carbohydrates broth was prepared separately. 5mL of the broth is dispensed into the tubes and Durham’s tubes are inserted in an inverted position into the tubes. Care should be taken to ensure that no air bubbles are trapped in the Durham’s tubes prior to the incubation. The tubes are plugged and sterilized under standard condition.

• Using sterile techniques, the tubes are inoculated separately and incubated at 370C for 24 hours. A control is maintained without inoculation.





• The tubes are observed for the fermentation of acids and production of gas.

SCREENING FOR FUNGI: (Harley Prescott, 2002, p-374-375)

• Autoclaved Potato Dextrose Agar and Martin Rose Bengal Agar (MRBA) were used for the isolation of fungi from tobacco samples.

• The samples obtained were first humidified for the enrichment of the culture.

• The sample was inoculated into the following media with the help of a sterile needle.

• The plates were left at 250C for 4-5 days. • The plates were inspected after 4-5 days to check

the growth.

• With the help of a sterile needle the fungal species were mounted on a slide, stained with lactophenol cotton blue, sheltered with cover slip and tapped gently.

• The genus was being identified by visual inspection in a light microscope at 40X magnification.

• Microscopic identification for the identification was conidial heads, Stipis, colour and length vesicle shape and serration covering, shape and conidial surface.

• The result was then matched with standard culture of Aspergillus sp.

RESULTS AND TABULATION

TRIAL 1: BRAND USED: Cigarette: Gold flake, Khaini: Madhu, Beedi: Lose piece available, Raw Tobacco: From processing market Hosur, Bangalore, Cigarette filter: Gold flake, Burning cigarette ashes: Gold Flake.

Table no 1: primary screening

Sample no.

Sample name Type of sample

Gram’s Character

Motility test

Oxidase test

Catalase test

Indole test

Methyl red test

VP test

1 Cigarette Type 1 + - - + - - + Type 2 - - - + + - +

2 Khaini Type 1 - - - + + - + Type 2 + - - + - - +

3 Beedi Type 1 + - - + - - + Type 2 + - - + - - +

4 Raw Tobacco Type 1 + - - + - - +

Type 2 + - - + - - +

5 Cigarette filter Type 1 + - - + - - + 6 Burning

Cigarette ashes No growth

NIL NIL NIL NIL NIL NIL NIL





Table no. 2: PRIMARY SCREENING: Colony characteristics:

Table no. 3: Physiological and biochemical characterization of organisms:

Table no. 4: Starch and Gelatin tests were performed for all the gram positive organisms.

Gram+ve colonies Starch test Gelatin liquification Cigarette-1 + +

Khaini-2 + + Beedi-1 + + Beedi-2 + +

Raw Tobacco-1 + + Raw Tobacco-2 + + Cigarette filter + +

Sample No.

Sample name Type of colonies

Size Margin Pigment Elevation Opacity

1. Cigarette Type-1 Small Smooth Non pigmented Raised Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

2. Khaini Type-1 Small Smooth Non pigmented Flat Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

3. Beedi Type-1 Pinpointed Smooth Non pigmented Flat Opaque Type-2 0.3cm Smooth Non pigmented Raised Opaque

4. Raw Tobacco Type-1 Pinpointed Smooth Non pigmented Raised Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

5. Cigarette filter Type-1 Pinpointed Smooth Non pigmented Raised Opaque

Sample Nitrate reduction test

Catalase from maltose plate Maltose Sucrose

Acid Gas Acid Gas Cigarette-1 + + + - + + Cigarette-2 + + - - + + Beedi-1 + + + - + + Beedi-2 + + + - + + Khaini-1 + + - - + + Khaini-2 + + + - + + Raw tobacco-1 + + + - + + Raw tobacco-2 + + + - + + Cigarette filter + + + - + +





Table no. 5: Lactose fermentation tests

Endospre Staining:

• Khaini-2, Raw tobacco-1 and Cigarette filter were positive for endospore staining. Terminal endospores are present in all the cases.

TRIAL 2:

BRAND USED: Cigarette: Gold flake, Khaini: Madhu, Beedi: Lose piece available, Raw Tobacco: From the processing market in Hosur, Bangalore, Cigarette filter: Gold flake, Burning cigarette ashes: Gold Flake

Table no. 6 PRIMARY SCREENING:

Sample No.


Gram’s Character

Motility test

Oxidase test

Catalase test

Indole Test

Methyl Red Test

VP Test

1. Cigarette Type-1 + - - + - - + Type-2 - - - + + - +

2. Khaini Type-1 - - - + + - + Type-2 + - - + - - +

3. Beedi Type-1 + - - + - - + Type-2 + - - + - - +

4. Raw Tobacco Type-1 + - - + - - + Type-2 + - - + - - +

5. Cigarette filter Type-1 + - - + - - + 6. Burning

cigarette ashes No

growth NIL NIL NIL NIL NIL NIL NIL

Table no. 7: Colony characteristics:

Culture Result on Macconkey Agar plate Lactose fermentation Cigarette-2 Pink colour colonies + Khaini-1 Pink colour colonies +

Sample No.


Size Margin Pigment Elevation Opacity

1 Cigarette Type-1 Small smooth Non pigmented Raised Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

2 Khaini Type-1 Small Smooth Non pigmented Flat Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

3 Beedi Type-1 Pinpointed Smooth Non pigmented Flat Opaque Type-2 0.4cm Smooth Non pigmented Raised Opaque

4 Raw Tobacco Type-1 Pinpointed Smooth Non pigmented Raised Opaque Type-2 Pinpointed Smooth Non pigmented Raised Opaque

5 Cigarette filter Type-1 Pinpointed Smooth Non pigmented Raised Opaque





Table no. 8: Physiological and biochemical characterization of organisms:

Table no. 9: Starch and Gelatin tests were performed for all the gram positive organisms.

Gram+ve colonies Starch test Gelatin liquification Cigarette-1 + + Khaini-2 + + Beedi-1 + + Beedi-2 + +


Table no. 10: Lactose fermentation tests

Endospre Staining:

• Khaini-2, Raw tobacco-1 and Cigarette filter were positive for endospore staining.Terminal endospores are present in all the cases.

Sample Nitrate reduction test

Catalase from maltose plate

Maltose Sucrose

Acid Gas Acid Gas Cigarette-1 + + + - + + Cigarette-2 + + - - + + Beedi-1 + + + - + + Beedi-2 + + + - + + Khaini-1 + + - - + + Khaini-2 + + + - + + Raw tobacco-1 + + + - + + Raw tobacco-2 + + + - + + Cigarette filter + + + - + +






TRIAL 3:

BRAND USED:Cigarette: Gold flake, Khaini: Madhu, Beedi: Lose piece, Raw Tobacco: From the processing market in Hosur, Bangalore, Cigarette filter: Gold flake, Burning cigarette ashes: Gold Flake

Table no. 11: PRIMARY SCREENING:

Sample No.


Gram’s character

Motility test

Oxidase test

Catalase Test

Indole Test

Methyl Red Test

VP Test

1 Cigarette Type-1 + - - + - - + Type-2 - - - + + - +

2 Khaini Type-1 - - - + + - + Type-2 + - - + - - +

3 Beedi Type-1 + - - + - - + Type-2 + - - + - - +

4 Raw Tobacco Type-1 + - - + - - + Type-2 + - - + - - +

5 Cigarette filter Type-1 + - - + - - + 6 Burning

cigarette ashes No growth NIL NIL NIL NIL NIL NIL NIL

Table no. 12: Colony characteristics:

Sample No. Sample name Type of colonies Size Margin Pigment Elevation Opacity 1 Cigarette Type-1 Small Smooth Non pigmented Raised Opaque

Type-2 Pinpointed Smooth Non pigmented Raised Opaque 2 Khaini Type-1 Small Smooth Non pigmented Flat Opaque

Type-2 Pinpointed Smooth Non pigmented Raised Opaque 3 Beedi Type-1 Pinpointed Smooth Non pigmented Flat Opaque

Type-2 0.6cm Smooth Non pigmented Raised Opaque 4 Raw Tobacco Type-1 Pinpointed Smooth Non pigmented Raised Opaque

Type-2 Pinpointed Smooth Non pigmented Raised Opaque 5 Cigarette filter Type-1 Pinpointed Smooth Non pigmented Raised Opaque

Table no.13: Physiological and biochemical characterization of organisms:

Sample Nitrate reduction test Catalase from maltose plate Maltose Sucrose Acid Gas Acid Gas

Cigarette-1 + + + - + + Cigarette-2 + + - - + +

Beedi-1 + + + - + + Beedi-2 + + + - + + Khaini-1 + + - - + + Khaini-2 + + + - + +

Raw tobacco-1 + + + - + + Raw tobacco-2 + + + - + + Cigarette filter + + + - + +





Table no.14: Starch and Gelatin tests were performed for all the gram positive organisms.

Gram+ve colonies Starch test Gelatin liquification Cigarette-1 + + Khaini-2 + + Beedi-1 + + Beedi-2 + +


Table no.15: Lactose fermentation tests


Endospre Staining:

• Khaini-2, Raw tobacco-1 and Cigarette filter were positive for endospore staining. Terminal endospores are present in all the cases.

Table no. 16: Tabulation for Fungus

Sample number

Fungus Size Stipiscolour Surface Vesicle Serration

Metula Covering

Shape Conidia surface

1 Aspergillus 4.5 cm Light brown Smooth Biseriate large size

Entirely Glubose Very rough, irregular





RESULT:

• The bacteria found were Klebsiella oxytoca and Bacillus. • Fungus found was Aspergillus sp.





DISCUSSION

In this study the presence of microflora in tobacco containing forms were to be detected. Tobacco smoking is the practice where tobacco is burnt and the resulting smoke is inhaled which causes various types of diseases and disorders. There are various disorders such as throat and lung cancers are known in case of practicing tobacco. But microbial diseases from tobacco practice are rarely well-known. This experiment with tobacco and its associated forms was carried out to isolate various bacterial and fungal strains from region of Hosur, Bangalore. This was carried out on the basis of staining and basic biochemical tests. There had been found presence of Klebsiella oxytoca (Gram negative bacteria) and Bacillus (Gram positive bacteria) species. Klebsiella oxytoca is a Gram negative bacterium which specifically gives indole test positive wherein other Klebsiella species are negative for indole. Cigarette (Type 2 type of colonies) and Khaini (Type 2 type of colonies) were found with Klebsiella oxytoca colonies. Klebsiella could be observed with naked eye with the observation of shiny layer on the colony plate due to presence of capsule. The confirmatory tests are carried out using Gram staining and different other biochemical tests. Klebsiella showed positive for Catalase, VP, Indole and Nitrate reduction test. It did not produce gas or acid in either in maltose fermentation test whereas acid and gas production was visualized on sucrose plates. Klebsiella was found negative for Methyl red and Oxidase tests. Klebsiella was cultured on Macconkey Agar plates which act as selective media for Gram negative, lactose fermentors. There was another species of bacteria found in the tobacco, Bacillus sp. which is a Gram positive bacterium. Bacillus gave positive results for Catalase, VP, Nitrate reduction tests and was found producing acid with no gas on maltose plates but there were appearance of both acid and gas on sucrose plates. Bacillus was also tested for Starch and Gelatin liquification and was positive for both. There was presence of terminal endospore in Bacillus species. Fungal species found Aspergillus was slightly brown in colour with smooth surface. The samples obtained were humidified and was inoculated in fungal media and left at incubation for 5-6 days. The organism was detected by

visual inspection under light microscope at 40X magnification. There was no presence of any thermophilic microflora in the ashes of burning cigarette. These microflora found in tobacco are not completely pathogenic and could be called opportunists. They can certainly cause infection but in immuno-compromised individuals.

CONCLUSION:

Smoking is the most common method of consuming tobacco, and tobacco is the most common substrate smoked. The agricultural product is often mixed with additives and then pyrolyzed. The resulting smoke is then inhaled and the active substances absorbed through the alveoli in the lungs. The active substance triggers chemical reactions in nerve ending, which heighten heart rate, alertness, and reaction time. Dopamine and endorphins are released which are often associated with pleasure. In most communities men are more likely to smoke than are women, though the gender gap tends to be less pronounced in lower age groups.

In conclusion, the present study has demonstrated that tobacco chewing can cause opportunistic infection to the immuno-compromised individuals. Gram negative Klebsiella oxytoca, Gram positive Bacillus and Aspergillus sp. of fungal strain were found from the samples collected, though there was no growth found from the ashes of burning cigarette. Thus it could be accomplished that other than physical disorders, tobacco chewing practice might cause microbial infections too.





REFERENCE:

Harley Prescott (2002). 5th edition- Laboratory exercises in Microbiology. p- 46,58-59,129,140,149,155-157,158, 166,170,180,374-375

Patterson F,Lerman C, Kaufmann VG, Neuner GA, Audrain-McGovern J. (2004).Cigarette smoking practices among American college students: review and future directions. 52(5):203-10.

Michael Rabinoff, Nicholas Caskey, Anthony Risslingand Candice Park(2007).Pharmacological and chemical effects of cigarette additives.Am J Public Health.97(11):1981–1991. Eng R, Smith SM (1991). The differential effect of cigarette smoke on the growth of bacteria found in humans.Ertel A, Chest. 100(3):628-30.

John L. Pauly and Geraldine Paszkiewicz. Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation.Journal of Oncology. Volume 2011 (2011), Article ID 819129, 13 pages.

Jeffrey D .Hasday, Rebecca Bascom (1999). Bacterial Endotoxin Is an Active Component of Cigarette Smoke. CHEST.115(3):829-835

R. P. Straka and J. L. Stokes (1957). Rapid Destruction of Bacteria in Commonly Used Diluents and Its Elimination.ApplMicrobiol. 5(1): 21–25. R. RYLANDER and P. HAGLIND(1984).Airborne endotoxins and humidifier disease.Clinical & Experimental Allergy.14(1) :109–112.

Citation of this article: Dona Saha, Anwesha Purkayastha, Prajesh (2004). ISOLATION AND IDENTIFICATION OF MICROFLORA IN TOBACCO AND ITS ASSOCIATED FORMS FROM

REGIONS OF HOSUR, BANGALORE. JOBB. 2(2): 68-79

Source of Support: Nil Conflict of Interest: None Declared





Research articleResearch articleResearch articleResearch article

FORMULATION OF BABY PRODUCT AND USING MORINGA OLIFERA SEED OIL

Kirti Bandarkar 1* 1 Assistant Manager, R & D Cosmetics, Sri Sri Ayurveda Trust, Bangalore, India *Corresponding Author email id: [email protected]

ABSTRACT Moringa oleifera seed oil acts as good antioxidant because of its tocopherol and unsaturated fatty acid content which is considered to be more powerful than vitamin C. It can be concluded that these liquid soap reduces irritation, dryness and rashes, which occurs due to addition of synthetic antioxidant in product as well as increases the skin softening and conditioning. No significant change in color, odour, pH was obtained at room temperature and fridge temperature (4oC) but the sample in oven showed changes in color, odour, pH and thus slightly lost its aesthetic appeal. Color and viscosity were directly proportional to the concentration of Moringa oleifera seed oil. It can be said that these liquid soap improves the skin softening and reduces skin problems by regular use.

Key words: Moringa oleifera, active detergent content, Staphylococcus aureus, drum stick seed oil _________________________________________________________________________________________________________

INTRODUCTION The prime requirement of any of the baby product is absolute safety. Great care should be taken that raw materials used are not only efficacious but also innocuous, with no incidence of irregularity in their toxicological profile. Only the purest grades of material should be selected and careful attention paid to the types and levels of any impurities that may be present. This is increasingly important with the correct use of antioxidant and preservative, with which low level of contamination in the raw material may be potentially harmful to a baby’s delicate skin. If any doubt is encountered over raw material selection, significant confidence can be gained by the use of materials which confirms to the requirements of the British pharmacopoeia (BP), European Pharmacopoeia (EP) or united State pharmacopoeia (USP). Specification must encourage tight control over the quality of material used,

such that confidence in the batch to batch consistency is high. The concentration of raw materials must be carefully chosen to eliminate all risks, even of minor skin reactions. The comedogenic nature of any material destined for use in any baby products must also be considered. The comedogenicity of material describes its potential to form comedons or spots. On the skin use of comedogenic materials should be avoided. Materials used should exhibit very low microbial counts and the absence of any microorganism that can be classified as pathogenic is mandatory. This is particularly relevant in the case of talc, starches, natural gums and plant extracts, where high levels of contamination by microorganism sometimes pathogenic can occur. Of particular importance is the quality of water used. The purity of this ingredient a major component in variety of product is often overlooked. It is one of the most important common problem in the finished product. Particularly those of microbial nature (Modern Technology of Science by NIIR Board,p-173).





RAW MATERIALS INCORPORATED IN FORMULATING A BABY WASHES

1) Stearic acid: CH3[(CH 2)16]COOH (Martindale’s

Extra Pharmacopoeia,p-1756; Harry’s Cosmeticology,p-707; Jellinck J.S., G.L. Fenton,1970,p-153)

Uses: Stearic acid used as an emulsifying and solubilizing agent also incorporated for its lubricating property. It is used in product to modify the consistency. Toxic action: Harmless Action: Innocuous 2) Cetyl Alcohol: CH3(CH2)14CH2OH (Modern Technology of Science by NIIR Board,p160-161) Uses: Cetyl alcohol is used in topical preparations for its soothing water absorption, stiffening and emulsifying property. It also provides smooth and velvety feel to product. It imparts a smooth texture to the skin so that widely used in cosmetics. Toxic action: Non toxic Dermatological action: easily absorbed by the skin and renders skin velvety, probably due to retaintation of cetyl alcohol with epidermis. 3) Lanolin: (Jellinck J.S., G.L. Fenton,1970,p-166) Lanolin is a purified anhydrous waxy, fatty compound manufactured from the fleece of the domesticated sheep. Ovies Aries Linn belonging to family Bovidae. Uses: lanolin is used in the formulation when mixed with suitable vegetable oil, it gives emollient cream which penetrates the skin and becomes soft. It can absorb about 30% of water. It gives a distinctive quality to the product; increasing absorption of active ingredient and maintaining a uniform consistency for the product under most climatic conditions. Toxic action: Non toxic Dermatological action: lanolin has affinity for skin and is quite sticky as well as being physically hygroscopic. 4) Glycerin: (Martindale’s Extra Pharmacopoeia,p-1711-1712; The Wealth of India – Raw Materials, vol. VI 426-429) One of the most valuable product knows by virtue of its solvent property. Glycerin is an osmotic agent with lubricating, humectants and effective skin moisturizing property. Uses: Glycerin is often used in topical preparations for its moisturizing, wetting and lubricating properties. Long term use of glycerin results in improves skin condition and

reduced transepidermal water loss, increased coefficient of friction, increased skin smoothness. Because when it is absorbed it gives hygroscopic action which may enhance the moisture retaintation. Toxic action: Non toxic Dermatological action: Skin becomes hygroscopic and soft without irritation. 5) Sodium Lauryl Sulphate: (Martindale’s Extra Pharmacopoeia,p-1534) Uses: sodium lauryl sulphate is an anionic emulsifying agent. It is detergent and wetting agent. Effective in both acidic and basic solution. It is used in skin cleansers. Toxic action: Non toxic Dermatological action: Less irritant. 6) Cocobetain: (Harry’s Cosmeticology, pg.441-442) It is having very good surfactant property. Uses: It is used as a good cleanser with high foaming property. They are mild and effective in nature; they are low irritant so that used in baby products. Toxic action: Non toxic Dermatologic action: Less irritant. 7) Coco mono ethanol amide: (Harry’s Cosmeticology, pg.441-442) The coco mono ethanol amide is an anionic surfactant. Uses: Coco mono ethanol amide is a very important body cleanser additive. They act as a viscosity modifier and give very well after effect. Since they exhibit softening properties they also improve volume and richness of lather. Toxic action: Non-toxic. Dermatological action: Good cleanser. 8) Methyl Paraben: C8H8O3 (Ralph G. Harry, 1963, p-287) It is the methyl ester of benzoic acid. Uses: It is water soluble preservative and used to prevent microbial contamination. Most effective functional concentration is 0.35 – 0.50%.

Toxic action: Non-toxic. 9) Propyl Paraben: C10H12O2 (Extra Pharmacopoeia, p-1287 and 6651) It is the propyl ester of benzoic acid. It is soluble in oil phase which is incorporated for its preservative action35. Uses: It is an antifungal preservative. It is used at a concentration of 0.25 – 0.50%. Toxic action: Non-toxic.





10) Water (Purified): Uses: water is used as a vehicle and solvent, it is of all cosmetic material the most compatible and completely non toxic. It is therefore irreplaceable when it is a question of

diluting other substances. It is also cheap and easily available. (Guenther and Guenther, p-141 and 161)

Moringa Oleifera (DRUMSTICK) SEED OIL. (Kirti Bandarkar, 2013) Family: Moringaceae Botanical Name: Moringa oleifera Popular Names: Moringa, Horse radish tree, Drumstick tree, Sahijan Parts used: Roots, Leaves, Bark, flowers and Seeds.

Table no. 1: Characteristic of Moringa oleifera seed oil

Table no. 2: Fatty acid composition of oil Properties:

� Moringa oleifera seed oil is most stable natural oil and found to be a good source of a behanic acid in nature.

� The oil produced is pale yellow in color, non-drying with a mild, characteristic nutty flavor.

� Perfume manufacturer esteem the oil for its great power of absorbing and retaining even the most fugitive odours and for its stability.

Characteristic Specific values

Specific gravity 0.912 – 0.915 Acid value 3.2 – 4 Saponification value 175 -186 Iodine vale 64.2 Unsaponificable matter 3.05% Density at 20o C 0.908 Viscosity at 20o 92.6 cps Melting point 21o C Smoke point 230o C

Fatty acids Percentage Myristic acid 0.1% Palmitic acid 9.3% Stearic acid 7.4% Behanic acid 8.6% Oleic acid 65.7% Palmitoleic acid 1.4% Linolic acid 1.5% Arachidic acid 3.7% Eicosenoic acid 2.3% Cerotic acid 1.3% Phytosterol 9%





� The skin moisturizing benefits are derived from the fact that Moringa oleifera seed oil is high in vitamin A and C and unsaturated fatty acids.

� Moringa oleifera seed oil contains antiseptic and anti-inflammatory properties, which help heal minor skin complaints such as cuts, burns, insect bites, rashes and scrapes quickly.

� Oil is highly unsaturated and it is liquid at room temperature with low melting point at 20.5oC

Uses:

� The oil is rich source of oleic acid which is useful in soap manufacturing and soothing of skin.

� The oil contains phytosterol which is act as a natural antioxidant.

� It is used as good absorbent. � It is very useful as a carrier and base oil.

FORMULATION AND DEVELOPMENT (Harry’s Cosmeticology, pg.111-113) Baby wash is undoubtedly the most popular bath preparation currently in the market and they have enjoyed a very healthy growth in recent years. While formulating the product following points were kept in mind.

1) It should provide copious foam at minimal detergent concentration.

2) The foam should be stable, particularly in the presence of soap and soil and with the wide limits of temperature.

3) It must be no-irritating to skin, eyes and mucous membrane.

4) It should have adequate detergent power so that it will cleanse the body efficiently. In order to counteract any excessive harshness to the skin it is advisable to include a low level of skin emollients.

5) Only small amount of the product should be required for the desired effects55.

Baby bath products or cleansing products mostly used over all the body, the baby skin is very delicate and sensitive. Therefore it is necessary to prepare a base as simple as possible containing non-irritant ingredients. These points were taken into consideration before formulating a base. Therefore it was decided to prepare a baby wash base using some fatty acids and sodium lauryl sulphate has the advantage of being relatively easy to formulate and have very less stability problems.

Base should give the fallowing requirements:

1) It must be compatible in every respect 2) As the vehicle for active ingredient it must be

able to dissolve them 3) The base should not hamper the effectiveness

of the active ingredient, but should enhance the same

4) From the consumer point of view it should be aesthetically appealing.





Table no.3 Selection of Base is as follows

Sl. no. Ingredients

F1 (%) F2 (%) F3 (%) Uses

A Water Phase 1. Sodium lauryl sulphate 30 25 25 Foaming agent

2. Distilled Water 52.7 55.1 55.65 Solvent

3. Glycerin 5 5 5 Humectant

B Oil Phase 4. Coco mono ethanol amide 2.5 2.5 3 Surfactant, foam booster

5. Coconut oil 1 1 1 Active

6. Stearic acid 5 4 4 Fatty acid

7. Lanolin 1 2 2 Emollient

8. Cocobetain 3 3 3 Foam booster, conditioner

9. Cetyl alcohol - 2 4 Viscosity modifier 10. Methyl Paraben 0.05 0.05 0.05 Preservative

11. Lavender oil 0.2 0.2 0.2 Perfume

Base F3: Modification: To increase the foaming power the percentage of foam booster was increased and to increase the consistency, the percentage of thickner i.e. cetyl alcohol was increased. Observation: Base 3 had good foaming and consistency, so base 3 (F3) was selected as a final formulation.The incorporation of active in selected base is shown in table no.4 which is as follows:

DEVELOPMENT AND SELECTION OF FORMULA

Table no. 4 Incorporation of Moringa oleifera seed oil in the selected base

Sl. no Ingredients

F4 (%) F5 (%) F6 (%) F7 (%) Uses

A Water Phase 1. Sodium lauryl sulphate 25 25 20 17.5 Foaming agent

2. Distilled Water 52.75 52.5 55 56.4 Solvent

3. Glycerin 5 5 8 8 Humectant

B Oil Phase

4. Coco mono ethanol amide 3 3 3 3 Surfactant, foam booster

5. Moringa Oleifera seed oil 1 2 2 2.5 Active 6. Stearic acid 4 4 4 4 Fatty acid

7. Lanolin 2 2 2 2.5 Emollient 8. Cocobetain 3 3 3 3 Foam booster, conditioner

9. Cetyl alcohol 4 3.5 3 3 Viscosity modifier

10. Methyl Paraben 0.05 0.05 0.05 0.05 Preservative

11. Lavender oil 0.1 0.1 0.3 0.3 Perfume





PROCEDURE OR METHOD OF PREPARATION: (Harry's Cosmeticology p-93-101).

� Sodium lauryl sulphate was soaked in a measured quantity of water. � Oil phase and water phase was weighed separately in beaker. � Sodium lauryl sulphate was added in water phase. � Both the phases were heated in a water bath up to 75o C. � Then water phase was added in oil phase and again heated it for 2 minutes. � After that mixture was transferred into mortar. � Mixture was triturated slowly. � When temperature goes down at 40o C, perfume was added. � Finally product was stored in proper container and tested for general appearance and stability.

Trial IV-F7 Modification: The amount of sodium lauryl sulphate was reduced; to give emoliency and aesthetic effect after wash, the percentage of lanolin and active was increased. Observation: The product was good in terms of foaming, consistency and emoliency, so trial F7 was selected as a final formulation. THE EXPERIMENTAL ANALYSIS OF FINISHED PRODUCT

The analysis of finished products was carried out by fallowing methods,

1) Physical method 2) Chemical method 3) Microbiological method

Physical Method

Appearance: As per the BIS standards of shampoo, the baby wash should be in the form of flowing type of the product.

pH: (IS 7884:1992 pg. 4-5)

Principle: The pH value conventionally represents the acidity or alkalinity of the solution. In the pharmacopeias the standard limits of the pH have been provided for those pharmacopoeia substance in which pH as a measure hydrogen ion concentration is important from the stand point of stability or physiological stability. Determination was carried out at temperature 25oC unless otherwise specified in the individual monograph. Apparatus: pH of the finished product was determined in the laboratory using pH Elico meter model L1-120, fitted with PPC type combined pH reference electrode.

Procedure: The pH of the finished product was determined as per Indian Pharmacopoeia. Observation: pH of baby wash at 25+2oC was found to be 6.8

Viscosity: (Indian Pharmacopoeia, vol. II: p-A-67)

Principle The viscosity is an expression of the resistance of the fluid to flow, higher the viscosity greater the resistance to flow the fluid. The viscosity of baby wash plays an important role in determining the products performance. Viscosity determines the feel and body of the product. Apparatus: Viscosity of the finished product was determined by using Brookfield viscometer (model-DV+). Procedure: The viscosity of the finished product was determined as per Indian Pharmacopoeia.





Chemical Method Determination of Active Detergent Content (IS 7884:1992 pg. 4-5) Principle:

When equivalent amount of cationic and anionic detergents are present in two phase mixture of water and chloroform, methylene blue will color two phase to same degree. Sodium alkyl benzone sulphonate and sodium lauryl sulphate or any other detergent can be titrated with standard solution of cetyl trimethyl ammonium bromide.

Procedure: The active detergent content of finished product was determined as per IS (Indian Standard) Shampoo, soap based – Specification

Calculation: Percent combined SO3 = V x n x 800/M. V – Volume in ml of solution A used in titration N – Normality of solution A M – Mass in gm of sample in the aliquot

% active detergent content = % combined SO3 x molecular mass of active detergent/ 80 V = 5.4 ml N = 0.00129N M = 1.603 gm % combined SO3 = 5.4 x 0.00129 x 800/1.603= 3.477% % active detergent content = % combined SO3 x molecular mass of active detergent/ 80 Molecular mass of the active detergent (SLS) = 340 % active detergent content = 3.477 x340/80 =14.777% Determination of Foam Height (IS 7884:1992 pg. 5-7) Principle: In order to check; the ability of baby wash to produces lather, the volume of the foam obtained under specific experimental condition is determined. Procedure: The foam height of finished product was determined as per IS (Indian Standard) Shampoo, soap based – Specification

Calculations: Foam height = (level of foam + sample solution) – level of only sample = V1 – V2

= 150 -7 = 133 mm

10 ml = 15 mm Foam height = 199.5mL

MICROBIAL METHOD

The microbial tests are designed for the estimation of the number of viable aerobic micro-organisms present and for detecting the presence of designated microbial species in substance. The main objective of carrying out the microbial analysis is to determine that the product body wash is within the limits according to the specification (TPC -1000 cfu / g: PATHOGENS – absent ). It should not cause any microbial infection to the skin, therefore the microbial analysis for TPC and PATHOGENS are carried out. In order to count the number of micro organisms in the body wash SOP was used and adapted for isolation.

1. Total plate count: (R. N. Bhattacharya, 1986) Aim: To determine the total plate count of baby wash. Equipment: Incubator, hot air oven, thermometer, autoclave. Requirement: Pipettes, petri dishes, dilution bottles, conical flasks, cotton, weighing balance. Media: Nutrient agar, Sabourataud dextrose agar Procedure: 1. A pre-weight amount of the sample approximately

1 – 10 gms was transferred to 9mL of water. Mix it thoroughly and let it stand for 5 min.

2. With the sterile pipettes 1mL of the above solution was dispensed into sterile plates.

3. Nutrient agar was transferred into one plate and Sabourataud dextrose agar was transferred into another plate by shaking gently.

4. After the agar solidifies the plates were inverted and were incubated at 33+2o C for 48h in the case of nutrient agar for bacterial enumeration and 28+2o C for 3-5 days in the case of Sabourataud dextrose agar.

5. Plates were then counted for the number of colonies formed in it with the help of colony counter.

6. Average was then taken out and total pate count was then calculated with dilution factor.





Formula: TPC = P1+P2 /2 X 90/W P1 – Total plate count for the count P2 - Total plate count for the second plate W – Weight of the sample taken Calculation: = (3 + 4) / 2 x 90 /1 = 3.5 x 90 / 1= 315 Conclusion: The baby wash passes the requirement for the total plate count as per BIS specification of shampoo. 2. Staphylococcus Aureus: Aim: Identification of Staphylococcus aureus in the baby wash. Procedure: 1. Selective enrichment:

• Under the asceptic condition add 1.0 g of pooled sample to 10 ml of enrichment broth containing tryptone (10.0 g), yeast extract (5.0 g), sodium pyruvate (10.0 g), sodium chloride (100.0 g), distilled or deionised water (upto 1000 ml), final pH at 250 C (7.4 + 0.2).

• It was incubated at 35 + 20 C for 12- 16 hours 2. Selective plating;

• Gram-positive cocci appearing in clusters were streaked on nutrient agar. It was then incubated at 33 + 20 C for 24 hours. It shows black colony.

• Colonies were transferred from the surface of the medium to individual tubes each containing 0.5 ml of mammalian plasma.

• Simultaneously coagulase positive and negative cultures and was incubated in a water bath at 370 C, this tubes was examined at 3 hour and at subsequently every 30 min, for upto 6 hours.

Identification: Gram stain – Staphylococcus aureus if appears should be produced as a gram positive. EVALUATION Evaluation of formulated product is necessary as it enable the cosmetologist to know whether his product possess the required qualities or not. Assessment of stability of baby wash: The final acceptance of product depends on stability, appearance and functionality of the packed products. The following stability parameters were studied for the products.

1. Color 2. Odour 3. pH 4. Viscosity

All the samples prepared were subjected to accelerated test conditions and were kept at room temperature, in oven at 50o C and in refrigerator at 4o C.

RESULT:

Physical method pH: The pH of the product was found to be within the limits as provided by the BIS specification of baby soap.

Viscosity: Observation and Calculation:

Spindle no. – 6 Rotations per minute – 100 Factor – 100 Dial reading – 55

Viscosity = dial reading x R.P.M. = 55 x 100 = 5500 cps

Observation: Viscosity of baby wash at 27+2oC was found to be 5500 cps. The viscosity of the product was found to be within the limits as provided by the BIS specification of shampoo.





Chemical method: 1) Active detergent content Observation: Active detergent content of baby wash was found to be 14.777%. The Active detergent content of the product was found to be within the limits as provided by the BIS specification of shampoo.

2) Foam height Observation: Foam height of baby wash was found to be 199.5 mm. The Foam height of the product was found to be within the limits as provided by the BIS specification of shampoo. The results of finished product as per BIS specification of shampoo is given in Table no. 5

Microbial method: The total plate count was found to be < 1000/gms. Staphylococcus aureus were found to be absent in the baby wash. Baby wash passes the requirement for the Staphylococcus aureus pathogens as per BIS specification of shampoo. REQUIREMENTS OF BABY WASH

Table no. 5 FINISHED PRODUCT

Sl.No. Characteristic Standards Results

1) Active detergent content percent by mass, Min. 5 14.78

2) pH at 27 + 2o C 5-9 6.8

3) Foam Height for one percent solution, min. 150mm 199.5mm

4) Color Light yellow Complies

5) Odour Floral (Lavender) Complies

6) Viscosity at 27 + 2o C 4000-8000 cps 5500cps

Stability results: Color change: The color change was observed visually with eyes. The color of all the baby wash samples kept in the fridge temperature (4oC), oven temperature (50oC), and room temperature were noted regularly at intervals of 2 days and the results are calculated as shown in table no. 8. Following is notation given to different color: OC: Original Color; VSOCC: Very slight original color change; SOCC: Slight original color change; OCC: Original color change





Table no. 6 Color change of baby wash sample kept at room temperature, fridge temperature (4oC) and oven temperature (50oC).

Sl.No. Days Room temp. Fridge (4oC) Oven (50o C) 1) 1 OC OC OC 2) 3 OC OC OC 3) 5 OC OC OC 4) 7 OC OC OC 5) 9 OC OC OC 6) 11 OC OC OC 7) 13 OC OC OC 8) 15 OC OC OC 9) 17 OC OC OC 10) 19 OC OC OC 11) 21 OC OC OC 12) 23 OC OC OC 13) 25 OC OC VSOCC 14) 27 OC OC VSOCC 15) 29 OC VSOCC SOCC 16) 30 OC VSOCC SOCC

GRAPH NO.1: Graphical representation of sample of baby wash which shows the color change at different temperature. RT- Room Temperature; OT- Oven Temperature; FT- Fridge Temperature

Odour Change: The odour change was noted simply by smelling the product. The odour of all the baby wash samples kept in the fridge temperature (4oC), oven temperature (50oC), and room temperature were noted regularly at intervals of 2 days and the results are calculated as shown in table no. 7 OO: Original Odour; VSOOC: Very slight original odour change; SOOC: Slight original odour change.





Table no. 7 Odour change of baby wash sample kept at room temperature, fridge temperature (4oC) and oven temperature (50oC) GRAPH NO.2: Graphical representation of sample of baby wash which shows the odour change at different temperature. RT- Room Temperature; OT- Oven Temperature; FT- Fridge Temperature

pH Changes pH was measured by using pH meter. The pH of all the baby wash samples kept in the fridge temperature (4oC), oven temperature (50oC), and room temperature were noted regularly at intervals of 3 days and the results are calculated as shown in table no.8

Sl.No. Days Room temp. Fridge (4oC) Oven (50o C)

1) 1 OO OO OO 2) 3 OO OO OO 3) 5 OO OO OO 4) 7 OO OO OO 5) 9 OO OO OO 6) 11 OO OO OO 7) 13 OO OO OO 8) 15 OO OO OO 9) 17 OO OO OO 10) 19 OO OO OO 11) 21 OO OO OO 12) 23 OO OO OO 13) 25 OO OO OO 14) 27 OO OO OO 15) 29 OO OO VSOOC 16) 30 OO OO VSOOC





Table no. 8 pH change of baby wash sample kept at room temperature, fridge temperature (4oC) and oven temperature (50oC)

Sl.No. Days Room temp. Fridge (4oC) Oven (50o C)

1) 1 6.8 6.8 6.8 2) 4 6.8 6.79 6.83 3) 7 6.8 6.79 6.83 4) 10 6.83 6.72 6.89 5) 13 6.87 6.68 6.92 6) 16 6.87 6.63 6.96 7) 19 6.95 6.67 6.99 8) 22 6.93 6.59 7.05 9) 25 6.97 6.53 7.09 10) 28 6.74 6.57 7.07 11) 30 6.76 6.58 7.08

Viscosity Changes The viscosity of gel was measured by using spindle no. 6 of Brookfield viscometer. And the viscosity change is shown in Table no.9

Table no. 9 Viscosity changes of baby wash sample at Room temperature.

Sl.No. Day Viscosity in cps 1) On the day of preparation of baby wash 5500 2) After 30 days 5900

GRAPH NO. 3: Graphical representation of sample of baby wash which shows the pH change at different temperature. OT- Oven temperature; FT- Fridge temperature; RT- Room temperature

Days





DISCUSSION:

The present study was carried out on Moringa oleifera seed oil to study its “Antioxidant” property in “baby wash” formulation. Moringa oleifera seed oil was analysed for its solubility, acid value, saponification value, iodine value and specific gravity. Moringa oleifera seed oil was incorporated in baby wash in different concentration and studied for stability followed by subjective evaluation. From the results obtained it can be concluded that; Moringa oleifera seed oil is easily soluble in all vegetable and mineral oils and mixed with number of organic solvents so it can be easily incorporated in different type of cosmetic preparations. In identification test, color reaction gives the positive results. Phytosterol was identified by IR (Infra Red) spectra; hence it is concluded that the Moringa oleifera seed oil passes the qualitative test. Results of study performed on antioxidant efficacy showed that as the concentration of oil increased, the percent decrease in oxidation was observed. Hence from this it is concluded that Moringa oleifera seed oil shows antioxidant property. Moringa Oleifera seed oil can be incorporated to the base formula up to 2.5% concentration.

CONCLUSION: Moringa oleifera seed oil being soluble in number of oils and organic solvent, so it can be easily incorporated in different cosmetic bases. Moringa oleifera seed oil can be tried with still higher safer concentration. Present formulation could be compared with other antioxidant formulations. Moringa oleifera seed oil contains large number of unsaturated fatty acids and can be incorporated in massage oils, massage creams and its efficacy can be carried out. Use of antimicrobial property of Moringa oleifera seed oil could be done in anti-acne cream, face washes and antidandruff hair oil.

The present work on Moringa oleifera seed oil for the antioxidant and miniaturization property could be further extended on the fallowing lines. Studies can be carried out to find out the optimum concentration of the Moringa oleifera seed oil required to get the desired efficacy. Moringa oleifera seed oil can be incorporated to aroma therapy for its good absorption property as carrier oil in the base and number of cosmetic products. Studies regarding

penetration and absorption of Moringa oleifera seed oil via skin can be studied. More stability parameters, toxicity and microbiological studies can be carried out in future. Subjective evaluation studies can be further extended in future.

REFERENCES: Extra Pharmacopoeia, 1982. 28th edition, p-1287 and 6651, 1290-v, 6665-e Guenther and Guenther, Essential oil p-141 and 161. Harry Ralph, J.B. Wilkinson; R.J. Moore; Harry’s Cosmeticology; 7th edition; published by George Godwin Longman; House Burnt Mill, Harlow, Essex, USA, p-93-101. Harry’s Cosmeticology, J.B. Wilkinson, R.J. Moore. 7th edition, published by George Godwin Longman. House Burnt Mill, Harlow, Essex, USA. p-111-113

Harry Ralph, J.B. Wilkinson; R.J. Moore. Harry’s Cosmeticology, 7th edition, published by George Godwin Longman. House Burnt Mill, Harlow, Essex, USA. P-287

Harry Ralph, J.B. Wilkinson, R.J. Moore. Harry’s Cosmeticology, 7th edition, published by George Godwin Longman. House Burnt Mill, Harlow, Essex, USA; p-441-442

Harry Ralph, J.B. Wilkinson; R.J. Moore; Harry’s Cosmeticology; 7th edition; published by George Godwin Longman; House Burnt Mill, Harlow, Essex, USA; p-707. Indian Pharmacopoeia,1996. Government of India. Ministry of Health and Family Welfare. vol. II; (p-z), published by the controller of publication, Delhi, p-A-67. IS (Indian Standard) Shampoo, soap based – Specification; IS 7884:1992, p- 4-5. IS (Indian Standard) Shampoo, soap based – Specification; IS 7884:1992, p-4-5. IS (Indian Standard) Shampoo, soap based – Specification; IS 7884:1992, p-5-7.





Martindale’s Extra Pharmacopoeia department of Pharmaceutical Science. 1st edition, published by Pharmaceutical Press, Landon, 1982, p-1534. Jellinck J.S., G.L. Fenton (1970). Skin and Hair. Formulation and Functions of Cosmetics. 1st edition, p-153. Jellinck J.S., G.L. Fenton (1970). Skin and Hair. Formulation and Functions of Cosmetics. 1st edition, p-166. Kirti Bandarkar (2013). EXTRACTION AND STANDARDIZATION OF MORINGA OLEIFERA SEED OIL. JOBB. 1(1): 15-20 Martindale’s Extra Pharmacopoeia department of Pharmaceutical Science. 1st edition, published by Pharmaceutical Press, Landon, 1982, p-1711-1712.

Martindale’s Extra Pharmacopoeia department of Pharmaceutical Science; 1st edition; published by Pharmaceutical Press; Landon; 1982; p-1756. Modern Technology of Science by NIIR Board; published by Asia Pacific Business Press; p-160-161 Modern Technology of Science by NIIR Board; published by Asia Pacific Business Press; p-173.

Ralph G. Harry. The Principle and Practice of modern Cosmetic Material, published by Bonard Hill (books) Ltd. Landon 287; p-1963. The Wealth of India – Raw Materials, vol. VI L-M Council of Scientific and industrial research, New Delhi, p-426-429.

Citation of this article: Kirti Bandarkar (2014). FORMULATION OF BABY PRODUCT AND USING MORINGA OLIFERA SEED OIL. JOBB. 2(2): 80-93

Source of Support:Nil Conflict of Interest: None Declared

Research articleResearch articlejobb.co.in/docs/vol2_issue2/all.pdf · The plant growth promotion activity of fermented leaf juice of Moringa oleifera (PKM-1) was determined by in-vitro

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