Introduction

Pathogenic and food spoilage bacteria have been considered as the primary causes of food-borne diseases and food quality deterioration in both developed and developing countries. In order to assure the food safety and to extend the shelf life of food products, additions of chemical preservative agents into food products or decontamination treatments via physical, chemical or biological process or their combinations have been widely applied in food industries ^[1]. Essential oils (EOs) can be extracted from various aromatic plants including herbs and spices as they are synthesized by these plants ^[2]. Spices and their EOs have been used as natural preservatives, to produce wholesome food products, for extension of shelf-life and to reduce pathogenic bacteria ^[3]. Cinnamon belongs to the Lauraceae family and the genus of Cinnamomum which comprises of about 250 species. Cinnamon is also a traditional herbal medicine that is widely distributed in China, India and Australia ^[4]. It has been applied in food, seasonings, cosmetics and medical industries because of its antimicrobial, antioxidant and anti-carcinogenic activities. The antimicrobial activity of cinnamon EO and its major composition had been previously evaluated .Our study aimed to investigate the antibacterial effect of cinnamon EO by incorporating in the sprouts to provide safety to consumer and better understanding on the use of natural antibacterial agents ^[5]. Sprouts present a unique food safety challenge compared to other fresh produce, as the sprouting process provides optimal conditions for the growth and proliferation of pathogenic bacteria. The sprout industry, regulatory agencies, and the academic community have been collaborating to improve the microbiological safety of raw sprouts, including the implementation of Good Manufacturing Practices (GMP), establishing guidelines for safe sprout production, and chemical disinfection of seed prior to sprouting. However, guidelines and best practices are only as good as their implementation ^[6]. The consumption of raw sprouts is considered high-risk, especially for young, elderly and immuno-compromised persons. By considering this all outbreaks we can conclude that there are lots of helpful ingredients and chemical which will reduce the microbial contamination. Addition of natural antibacterial agent to sprouts will help to eliminate the contamination to acceptable level. To reduce such risk at beginning level is difficult task, and to maintain the physical appearance and organoleptic properties also, so use of natural antibacterial agent will help to reduce contamination and will not deteriorate its physical appearance and organoleptic findings also ^[7].

Table 1: Antibacterial Assay

Materials and Methods

Table 2: Different Parameters Of Germination

Green gram sprouts

Green gram seeds were purchased from local super market, Potheri, Chennai, India Bacteria and Culture Conditions

E.coli used in this study was isolated from different water samples.

Essential oil

Cinnamon oil was purchased from local shop in Chennai, India

Table 3: Total Plate Count Of Sprouts Of 24 Hours

Antibacterial assay

The antibacterial activity of Cinnamonaldehyde was evaluated by disc diffusion method. The exponential phase cultures of E.coli were adjusted to the concentration of 1.02×10⁸ CFU/ml and were swabbed on Mueller Hinton Agar (MHA) plates. Sterile paper discs (6mm diameter) were loaded with 20μl of different of Cinnamonaldehyde, Sterile antibiotic discs were placed on MH agar and incubated at 37°C for overnight. Zone of clearance surrounding the discs was measured using a transparent ruler and the diameter was recorded in mm. Tween 80 was used as control. Values are described as mean ± SD of assays performed in triplicate

Minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC)

The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) was measured by broth dilution method using Mueller Hinton Broth (MHB). Overnight broth cultures of E.coli were adjusted. 20μl of different concentrations of Cinnamonaldehyde was placed in sterilized test tubes to which contain 100μl of overnight cultured broth. The tubes were incubated at 37°C for 18–24 h and OD600 values of the cultures were measured, and the lowest concentration that inhibited the bacterial growth was taken as the MIC; the determinations were performed in triplicates. All the MIC tubes (100μlof culture from each tube) were then used for spreading on Muller Hinton Agar plates for colony counting. The concentration at which no growth was observed was determined as minimal bactericidal concentration (MBC).

Total plate count

The total plate count TPC (aerobic, mesophilic organisms) defines how many aerobic (oxygen-loving), mesophilic (moderate-temperature-loving) micro-organism colonies such as bacteria, yeast and mould fungi will grow in 72 hours on an agar plate that was normed for microbiological testing at a controlled temperature of 30°C. The sample is disintegrated in a standard diluent. The fibre suspension is transferred and incubated. After the incubation time, the numbers of bacterial colonies are counted on the respective plates. The results are expressed separately as the total colony number per gram dry mass of the sample.

Determination of fat and protein content

 Analyses of protein, fat, were performed for three replicates. Methods used for the estimation were AOAC standard methods [28].

Color Analysis of Sprouts

The colour parameter of sprouts was monitored by Hunter L*, a* and b* values using Hunter Colorimeter. L* (lightness), a* (redness) and b* (yellowness) values were measured for sprouts in three replicates.

Table 4: Biochemical And Nutritional Analysis Of Control Samples (Green Gram Sprout)

Preparation of sprouts

Green gram seeds were purchased from local super market; sprouts were prepared at home at ambient temperature by soaking seeds in three times of water of its seed weight and soaked it for 6-8 hours. Though seeds were small in size it will take less time for soaking. After the proper soaking of seeds, water was drained from seeds. The drained seeds were kept in muslin cloth or sieve and covered using cloth for proper germination for 10-12hrs. Actually germination procedure varies with climatic conditions, as project was undertaken in Chennai due to humid climate seeds were sprouted in open climate without covering it with cloth as it was growing sticky and sour. Then sprouts were kept in water containing cinnamon essential oil prior to using for decontamination.

Table 5: Biochemical And Nutritional Analysis Of Sample Containing Cinnamon Oil

Sensory Analysis

The sensory analysis for various samples was conducted for taste, aroma, texture and appearance. The sensory evaluations were conducted on nine point hedonic scale. The panelists were asked to rate the acceptability of the product on a scale of 9 points ranging from 9 to be “like extremely” to 1 to be “dislike extremely”.

Sensory was carried out in between semi trained and untrained people for acceptability of product.

Result and Discussion

Isolation of E.coli from water samples

Escherichia coli contamination of water has emerged as an important public health concern. Drinking and recreational waters have been linked to human cases of disease Furthermore, E. coli can be present in every water source which can causes human illness, so E.coli was isolated from drinking water source which was used for germination of seeds, where total plate count was found for E.coli was 9.2 x 10⁸ CFU.

Table 6: Color Analysis Of Green Gram Sprouts After Addition Of Antibacterial Agents

Antimicrobial assay

Antimicrobial assay was carried out by Disk diffusion method to find out zone of inhibition diameter for cinnamon essential oil purchased from market.

Germination of green gram

Sprouting is the practice of germinating seeds to be eaten raw or cooked. Sprouts can be germinated at home or produced industrially. They are a prominent ingredient of the raw food diet, some parameters to be maintained while germination.

Effect of Antibacterial agent on sprouts  after incorporation of essential oil 

Cinnamon essential oil was incorporated in water which used  by mixing it with tween 80 for reduction of E.coli contamination as pretreatment for soaking of sprouts and Antibacterial effect study was carried out by doing total plate count after some hours interval.

Effect of antibacterial agent on Biochemical and nutritional content of sprouts.

Green gram seeds purchased from market and sprouted and analyzed for protein and fat which shows 3.2g, 0.2g protein and fat respectively, Non significance was observed in content of sprouts after addition of antibacterial agent than control samples.

Color analysis of green gram sprouts by hunter colorimeter

The values of color analysis of green gram sprouts after addition of antibacterial agent showed that it has the same color as like control. It shows there is no significant color difference between control and cinnamon oil containing samples.

Sensory analysis

Sensory evaluation was carried out using 9 point hedonic scale. The product was optimized based on sensory analysis with acceptable score 8.5. The test samples were reasonably acceptable .Sensory analysis of the control sample and the test samples (sprouts pre treated with cinnamon (EO) essential oil) was conducted. The test sample sprouts contained 1%, 1.8%, 2.5%, 3.5% ,4% EO  water for pretreatment   respectively. Subjects were asked to sample the sprouts and scores were given based on taste, texture, appearance, aroma and overall acceptability. The sensory analysis result showed that the samples which treated with 2.5% of Cinnamon oil among the other oil incorporated sprouts scored more on the analysis. The overall acceptability of the samples containing 2.5% oil was considerably higher than the other test samples and the control.

Table 7: Sensory Evaluation

Conclusion

Sprout is the intermediate stage between the seed and plant containing cotyledon having great kind of nutritional contents. It is said that sprouts ‘represent the miracle of birth’. They are in the true sense, super foods. They are alkaline, whole, pure, and natural foods. It is inexcusable that though aware of their miraculous effects, sprouts are usually consumed in raw and cooked form, and which cause the chances of human illness due to presence of E.coli which came from water source used for germination of seeds. Aim of this project is that to reduce the contamination level to the acceptable level by giving pretreatment of natural antibacterial agent i.e. Cinnamon essential oil. Sprouts are the protein rich products which are used as energy boost, which gives proper nutrition to human and that nutrition was not affected by cinnamon essential oil, so this project indicates that contamination of microorganism can be reduced by some natural potential agents and that not cause any significant effect on quality parameters of sprouts.

Acknowledgement

The authors are thankful to the Dr. K.A. Athmaselvi, HOD, Food Process Engineering Department and the Dr. M. Vairamani, Dean, School of Bioengineering, SRM University, for providing necessary facilities and continuous support to carry out the research work.

References

1. Brul S, Coote P. Preservative agents in foods: mode of action and microbial resistance mechanisms. J. Food Microbiol.1999;50:1–17
2. M. Tajkarimi, S.A. Ibrahim Antimicrobial herb and spice compounds in food, Food Microbiology and Biotechnology Laboratory, North Carolina A&T State University, 171-B Carver Hall, Greensboro, NC 27411-1064, USA
3. Sara Burt* Essential oils: their antibacterial properties and potential applications in foods by Department of Public Health and Food Safety, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80175, 3508 TD Utrecht, The Netherlands
4. Jayaprakasha, G.k. and Jagan Mohan Rao, L(2011) chemistry , Biogenesis and Biological Activities of Cinnamomum Zeylanicum. Critical Review in Food Science and Nutrition,5.pp 547-562
5. Akbas MY, Olmez H ,Inactivation of Escherichia coli and Listeria monocytogenes on iceberg lettuce by dip wash treatments with organic acids 2007 Jun;44(6):619-24.
6. G, Patil U, Chavan J K, Kadam S S, Salunkhe D K. 1993.”Effects of dry heat treatments to peanut kernels on the functional properties of the defatted meal.” Plant foods for human nutrition (Dordrecht, Netherlands) 43 (2): 157-62.
7. Pandima Devi∗, S. Arif Nisha, Sakthivel, S. Karutha Pandian R. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane Department of Biotechnology, Alagappa University, K.araikudi 630 003, Tamil Nadu, India
8. Stelios Viazis, Mastura Akhtar, Joellen Feirtag, Francisco Diez-Gonzalez* Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave. St. Paul, MN 55108, USA
9. A. Ayoola1*, F. M. Lawore1, T. Adelowotan2, I. E. Aibinu2, E. Adenipekun2, H. A. B. Coker1 and T. O. Odugbemi Chemical analysis and antimicrobial activity of the essential oil of Syzigium aromaticum (clove) Depatment of Medical Microbiology and Parasitology, College of Medicine, University of Lagos, Lagos, Nigeria.
10. Jeongmok Kim, Maurice R. Marshall, and Cheng-i Wei * Antibacterial Activity of Some Essential Oil Components against Five Foodborne Pathogens Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 3261 1-0370.
11. National Advisory Committee on Microbial Criteria for Foods (NACMCF). Food and Drug Administration., 1999, Microbiological safety evaluations and recommendations on sprouted seeds. International Journal of Food Microbiology 52, pp. 123-153
12. Patterson, J.E., Woodburn, M.J. (1980). Klebsiella and other bacteria on alfalfa and bean sprouts at the retail level. Journal of Food Science, 45, pp. 492-495 Peńas, E., Gómez, R., Frías, J., Vidal-Valverde, C., 2008, Application of high pressure treatment on alfalfa (Medicago sativa) and mung bean (Vigna radiata) seeds to enhance the microbial safety of their sprouts. Food Control, 19, pp. 698-705
13. Piernas, V., Guiraud, J.P., 1997, Disinfection of rice seeds prior to sprouting.Journal of Food Science, 62, pp. 611-615
14. Prokopowich, D., Blank, G., 1991, Microbiological evaluation of vegetable sprouts and seeds. Journal of Food Protection, 54, pp. 560-562
15. Randazzo, C.L., Scifò, G.O., Tomaselli, F., Caggia, C., 2009, Polyphasic characterization of bacterial community in fresh cut salads. International Journal of Food Microbiology, 128, pp. 484-490
16. Robertson, L.J., Johannessen, G.S., Gjerde, B.K., Loncarevic, S., 2002, Microbiological analysis of seed sprouts in Norway. International Journal of Food Microbiology, 75, pp. 119-126
17. Soylemez, G., Brashears, M.M., Smith, D.A., Cuppett, S.L., 2001, Microbial quality of alfalfa seeds and sprouts after a chlorine treatment and packaging modifications. Journal of Food Science, 66, pp. 153-157
18. Splittstoesser, D.F., Queale, D.T., Andaloro, B.W., 1983, The microbiology of vegetable sprouts during commercial production. Journal of Food Safety, 5, pp.79-86
19. Taormina, P.J., Beuchat, L.R., Slutsker, L., 1999, Infections associated with eating seed sprouts: an international concern. Emerging Infectious Disease, 5, pp. 626-634
20. Viswanathan, P., Kaur, R., 2001, Prevalence and growth of pathogens on salad vegetables, fruits and sprouts. International Journal of Hygiene and Environmental Health, 203, pp. 205-213
21. Weiss, A., Hertel, C., Grothe, S., Ha, D., Hammes, W.P., 2007, Characterization of the cultivable microbiota of sprouts and their potential for application as protective cultures. Systematic and Applied Microbiology, 30, pp. 483-493
22. EI-Beltagy AE (1996) Effect of Home Traditional Methods on Quality Aspects of Some MSc Thesis, Faculty of Agriculture, Menofyia University, Shibin El-Kom, Egypt.
23. Alonso R, Orúe E, Marzo F (1998) Effect of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds. Food Chem 63:505–512.
24. Abu-Samaha OR (1983) Chemical, Technological and Nutritional Studies on Lentil. MSc Thesis, Faculty of Agriculture, Alexandria University, Egypt.
25. El-Adawy TA (1986) Chemical and Technological Studies on Faba Bean Seeds. MSc Thesis, Faculty of Agriculture, Menofiya University, Egypt.
26. Hulse JH, Rachie KO, Billingsley LW (1977) Nutritional Standards and Methods of Evaluation for Food Legume Breeders. International Center for Agricultural. Research in Dry Areas (ICARDA). Ottawa, Canada, KIG. 3Hg.
27. AOAC (1990) Official Methods of Analysis, 14th ed. Washington DC: Association of Official Agricultural Chemists. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356