ACKNOWLEDGE I am very much thankful to my guide Dr.

Somya Mehra, Lecturer, IBMER, Manglayatan University who guided me at every step involved in my project. I am grateful to have such a supporting hand besides me CONTENTS 1. INTRODUCTION 2. REWIEW 3. MATERIAL AND METHOD 4. RESULTS 5. DISCUSSION 6. SUMMERY PAGE NO.

IntroductionFruit normally means the freshly seed associated structure of certain plants that are sweet and edible in the raw state such as apple, orange, grapes, banana etc. Fruit juice is any substance that is consumed to provide nutrients support for the body. It is usually of plant or animal origin and contained essential nutrients, such as carbohydrates, fats, proteins.vitamines or minerals. The substance is ingested by an organism and assimilated by the organism cells in an effort to produce energy maintain life and growth .most of the energy consumed by the world population is supplied by the food industry which incorporation by multination corporations. Fruit are generally high in fiber, water, vitamin C and sugar although this latter varies widely from traces as in lime to 61% of the fresh weight of the date, fruit also contain various phytochemicals. Under most nutrition factsheet and which research indicates are required for proper long term cellular health and disease prevention regular consumption of fruit is associated with reduced risks of cancer, cardiovascular disease (especially coronary heart disease) stroke, Alzheimer disease and some of the functional declines associated with aging. Some scientist research on packed fruit juice and observed that microbial contamination in the packed juice which is due to some condition (pH, moisture, not proper handling , preservative etc.) The preservation of the juice was carried out using sugar, benzoic acid, citric and a combination of citric and benzoic acid under room temperature. Due to contamination the microbes (E.coli, C. albicans) found in packed fruit juice and these are caused disease (diarrhea, gastroantitis, meningitis in infants, urinary track infection) . In 2002 -15% , and 2005- 23% people die. To investigation of microbial load in packed juice. Therefore from the above view a study is planned to investigate the microbial load/ contamination over the commercial available fruit juice. Therefore, by keeping the above fact, a study was planned to to investigate the microbial load in various fruit juice product which are commercial available we take sample eg. Apple juice , orange juice. We take Different sample of fruit juice (apple, orange) would be used for study.
Introduction:-

In a hot country like India, in general, the people will use refreshing and thirst quenching fruit juices. The products like fruit juices, squashes, cordials, crushes, syrups have been introduced on a commercial scale during the last few decades. Establishing a small scale unit based on the fruits available during the respective seasons will be both cost effective and qualitative.

Market:With the changing pace of human activity and changing life styles, the readymade fruit juices is also increasing. The product has growing market in all tourist centres, pilgrim centres, hotels & restaurants apart form household consumption. The household market is also growing in the nearest past.

Manufacturing Process:Fruits are peeled and made into small pieces. The juice is made by churning and blending in a mixer or crusher. Sugar, spices and ice is added to enhance the taste.

Review:1. Clement Jackson et al (2010) studied the bioload on the six branded fruit juices. They performed their experiment in three different phases namely saline solution, distill water and concentrated juice sample having positive control (culture containing E.coli and Candida albicans) and negative control. They observed that there was no viable microbial load in the sample having pH less than 4. But according to them, there might be dead microbes in the sample juice. 2. K agar, a novel isolation medium developed for the food industry, was compared with other
acidified media for isolation of Alicyclobacillus acidoterrestris spores. Spores were inoculated into apple juice, orange juice, and a fruit juice blend and then isolated on the following media: K agar, pH 3.7; semi-synthetic medium, pH 4.0; orange serum agar, pH 3.5; and minimal salts medium, pH 4.0. Media were incubated at 24, 35, 43, and 55 degrees C. Highest recovery of spores was obtained with either K agar or semi-synthetic medium, incubated at 43 degrees C.

3. Yeh JY et al (2004) studied on the Calcium lactate is used by the beverage industry as a source of
calcium to fortify fruit juice. The objective of this study was to evaluate the influence of various concentrations of calcium lactate on the fate of pathogenic and spoilage microorganisms in orange juice. Commercial nonfortified orange juice was supplemented with calcium lactate at a

concentration equivalent to 0, 5, 10, 15, 20, 25, or 30% dietary reference intake. The pH of each fortified juice was adjusted to 3.6 or 4.1. The prepared juice samples were inoculated separately with a three-strain mixture of salmonellae, a three-strain mixture of spoilage yeasts, and three single strains of spoilage bacteria including Alicyclobacillus acidoterrestris, Lactobacillus plantarum, and Lactobacillus sake. The contaminated juice was stored at 4 and 10 degrees C, respectively, for 6 to 7 weeks and assayed once a week for populations of salmonellae, spoilage yeasts, or spoilage bacteria. The results indicated that A. acidoterrestris was inhibited in all juice stored at 4 degrees C and low-pH juice stored at 10 degrees C. The bacterium, however, was able to grow at 10 degrees C in the high-pH juice with calcium lactate concentrations equivalent to 0 and 5% dietary reference intake.

4. Two aciduric, aerobic, sporeforming bacteria were isolated from pasteurized juices. The grampositive, catalase-positive rods produced spores that were located subterminally in a swollen sporangium. The cultures had an optimal pH of 3.5-4.0 for growth and preferred potato dextrose agar over many of the rich media usually used for cultivating sporeforming bacteria. Spore inocula grew well in apple juice and white grape juice. Red grape juice was inhibitory, perhaps because of the concentrations of certain phenolic compounds. The spores were sufficiently heat resistant to survive commercial pasteurization processes.

5. Tambekar D.H et al (2009) studies the highest bacterial contamination was observed in sweet
lemon (35%), pineapple (29%), pomegranate, apple, orange each with (12%) and mix fruit with a strain of bacteria showed (100%) contamination. Sweet lemon and pineapple juices were highly contaminated because it is maximally consumed and already peeled out quite before the juice preparation. In both the juices E.coli (40%) and P. aeruginosa (22%) were dominant organisms.

Material and Method:1. 2. 3. 4. 5. 6. 7.

Nutrient media Nutrient broth media Maconkay media Methyl red and voges media Laminar air flow cabinet located in a clean room. Sterile lab coat, caps, gloves Sterile cotton-plugged, spatulas, transfer pipettes (for liquid and semi-liquid products). 8. 70% alcohol.

9. Gram stain solutions 10. Light microscope 11. Sterile petri plate, beakers, test tubes, brown paper, tissue paper

12. Colony counter

Method:-

We select the Different sample of fruit juice (apple, orange) would be used for study for investigation of microbial contamination in packed fruit juice. We prepared the nutrients agar media in conical flask and autoclaved media. pouring the media in sterile perti plate in laminar flow hood and solidifing rest of the end. After we use standard method or the experiment so perform experiment in three phases A,B,C. In A phase we take 1l sample directly in the sample in laminar flow hood and spread the sample with the help of sterile loop. In B phase we used the serial dilution. In this phase diluted by the distilled water upto 104 and used sample for spreading 104 In C phase we used saline solution (0.76%) for dilution. After Incubated at 370 c for 24-48 hrs. inoculation these plates

Counting the isolated colony by using colony counter and pick the colony for staining. Biochemical Tests:1. Gram Staining 2. Catalase test 3. Methyl red 4. Voges-proskauer

STAINING METHOD

Prepare heat fix bacterial smear.

Flood the bacterial smear with Crystal violet (1 minute).

Wash it with distilled water.

Flood it with Grams iodine solution (1 minute)

Wash it with distilled water.

Flood it with 70% alcohol.

Rinse it with distilled water.

Flood it with safranine (30 seconds)

Rinse it with distilled water s .Allow it to dry.

Fix it with oil immersion

Observe under microscope.

CATALASE TEST Take a glass slide and wash it.

Put a drop of saline solution of bacterial culture on a glass slide and mix it gentely

Add 3-4 drops of hydrogen peroxide on it.

Look for vigorous bubbling occurring within 10 seconds.

METHYL RED

Prepare Methyl Red and Voges-Proskauer broth

MR-VP broth.

Add 0.5ml bacterial culture in 5ml broth containing test tubes.

Incubate at 370C for 48 hours.

After incubation add 5 drops of methyl red indicator to the test tubes.

Observe change in color

If they change colour it maen the result is positive VIGES PROSKAUER Take 0.5ml bacterial culture in 5ml MR-VP broth.

Incubate at 370C for 48 hours.

Add 12 drops of alpha-naphthol and 6 drops of potassium hydroxide

to it.

Wait for few minutes to develop colour at or near the surface.

RESULT:Table 1: Results of various juice samples after testing s/no. Sample Bacterial load cells/ml
4X103 cells/ml No growth observed

Suspected microorganisms
Stapphylococcous -

Morphological tests
Gram +ve, cocci -

1 2

Apple orange

Apple

Orange

CONCLUSION:
It can be concluded from above study that Commercial available packed fruits juices are also contaminated by several microorganisms which can be pathogenic and may be harmful for consumer. In our result out of two samples, one sample found heavy microbial load although it was preserved by using pasteurization method. However, from the results, it has been observed that the packed fruit juices are also not free from microorganism and is not found safe for consumer REFERENCES Chang S, Kang D (2004). Alicyclobacillus spp. in the fruit juice Industry: History, Characteristics and Current Isolation/Detection Procedures. Crit. Rev. Microbiol., 30: 55-74. Jay JM (1998). Intrinsic and Extrinsic Parameters of Foods that affect Microbial Growth. In: Modern Food Microbiology, 5th ed. Chapman and Hah., New York, pp. 354-355. Splittstoesser DF, Churey JJ, Lee CY (1994). Growth characteristics of

aciduric spore-forming bacilli isolated from fruit juices. J. Food Prot., 57: 1080-1083. Walls I, Chuyate R (2000). Isolation of Alicyclobacillus acidoterrstris from fruits juices. J. AOAC Int., 83: 1115-1120. Yeh JY, Ellis H, Chen J (2004). Influence of calcium lactate on the fate of spoilage and pathogenic microorganisms in orange juice. J. Food Prot., 67: 1429-1433..