TITLE: Method of preparing natural preservatives for shelf life extension
FIELD OF THE INVENTION
The present invention relates to the methods for preparing -preservative, more -particularly to natural preservatives for increasing the shelf life of the products.
BACKGROUND OF THE INVENTION
The demand for fruit juices has been increasing due to their health benefits. Due to the changes in social habits and dietary intake people prefer to consume RTS (Ready - To - Serve) beverages. Were these RTS beverages are preserved with chemical preservatives have led to increase in disease outbreaks linked mainly to fresh fruit juices in recent years. So the main aim of this work is to replace the role of chemical preservatives in fruit juices with natural preservative and thermally processed method.
The natural preservatives used in the shelf life extension are found to be more effective and it inhibits the growth of microorganisms. These natural preservative has no toxic effect and increases more health beneficial advantages to the consumers. People do started preferring to consume herbal and natural products rather than toxic chemical products.
In one of the prior art Japanese Patent Application Number JP6903656B2 titled Natural preservatives, antimicrobial agents, and compositions thereof discloses compositions and related methods that can act as preservatives, antimicrobial agents, or combinations thereof. A conceivable embodiment is of Albizia amara seeds and Magnolia officinalis, which can be used to preservative foods, cosmetics, dietary supplements, oral hygiene products and pharmaceuticals. Regarding preservatives derived from extracts derived from the bark.

In another prior art Chinese Patent application number CN103416480B titled Compound navel orange preservative prepared from plant extract discloses a compound navel orange preservative prepared from plant extract which is prepared by combining plant extract, food preservative, stabilizer and acidity regulator. The invention aims to replace pesticide bactericide, which is adopted as antibacterial agent during the storage period of navel oranges in China all the time, with the compound navel orange preservative, so as to solve the problems of food safety and environment pollution caused by pesticide application. The main technical characteristics of the compound navel orange preservative are as follows: the principal components of the preservative comprise 10-15% of a magnolia officinalis extract, 20-30% of a clove extract, 10-20% of potassium sorbate, 10-20% of propylparaben, 2-5% of citric acid and 1-5% of stabilizer. The compound navel orange preservative is brown emulsion. When in use, the compound navel orange preservative is diluted with water, wherein the volume of the diluted compound navel orange preservative is 20 times of that of the compound navel orange preservative before dilution; navel oranges are soaked in the compound navel orange preservative for 30 seconds; after being subjected to natural air drying, the navel oranges are stored or subjected to postharvest commercialization treatment. The compound navel orange preservative can efficiently lower the rotting rate of the navel oranges, and delay the quality losses of the navel oranges during the storage and fresh-preservation period of the navel oranges. The compound navel orange preservative can also be applicable to the postharvest storage and fresh-preservation of other fruits and is particularly suitable for the storage and corrosion protection of citrus fruits.
In yet another prior art US patent application number US20200360260A1 titled Topical composition comprising antimicrobial lipid discloses a topical composition comprising an antimicrobial lipid found in the sebum or stratum corneum of human beings, other than saturated Cg to is fatty acids, wherein said composition further comprises a biphenol obtainable from Magnolia spp. Also disclosed is a method of

providing topical antimicrobial benefit comprising a step of applying a safe and effective amount of the topical antimicrobial composition.
The present invention has overcome the.drawbacks in the prior art, wherein natural preservatives were added to increase the shelf life period of fruit juices. Pineapple juice and grape juice samples were preserved with natural preservatives and was stored for a period of 90 days. These fruit juices had no physiochemical or biological changes.
OBJECT OF THE INVENTION
Accordingly, the primary object of the present invention is to preserve both the pineapple juice and grape juice using natural preservatives and thermally processing methods.
1. The primary object of the present invention is to reduce the microbial growth in
the both juices using natural preservatives.
2. It is another object of the present invention is to make a product and replace the
chemically preserved commercial products with naturally preserved products.
SUMMARY OF THE INVENTION
It will be understood that this disclosure is not limited to the particular methodologies described, as there can be multiple possible embodiments of the present disclosure which are not expressly illustrated in the present disclosure. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present disclosure.

The present invention relates to a method of preparing natural preservatives for shelf life extension.
It is an aspect of the present invention to extract crude natural preservative, wherein said natural preservative are prepared by extracting juice from lime, ginger, indian borage, indian gooseberry, clove and cinnamon thereby preparing the natural preservatives;
It is another aspect of the present invention, wherein the lime extract is prepared by squeezing the lemon, wherein the ginger extract is prepared by grating and squeezing the ginger and filtering it, wherein the indian borage extract is prepared by rinsing the leaves, and grinding the leaves to prepare a paste and filtering it using filter paper, wherein the indian gooseberry extract is prepared by cutting the fruit and grinding it and further extracting the filter using filter paper, wherein the clove extract is prepared by grinding the clove to fine powder and mixing 0.25g of clove and add 25ml of distil water and allowing it to boil 80 V. for lOmin and further the aqueous solvent was filtered using the filter paper and wherein the cinnamon extract is prepared by dissolving 0.5g of cinnamon powder in 5ml of distil water and boiled at 80"C for lOmin and further the aqueous solvent was filtered using the filter paper.
It is yet another aspect of the present invention to preserve the fruit juice by means of natural preservatives, wherein the said pineapple juice is centrifuged and supernatant is filtered and the said filtered is mixed with distilled water at dilution rate of 70% juice ; 30% water for pineapple juice and 60% Juice: 40 %water for grape juice and preservatives at various concentration of 3%, 1% and 7% is added fnr pineapple juice and concentration of 3%, 1%, 0.5%, 0.2% and 5% is added for grape juice and the mixture is boiled at 80°C for 2 minutes and further autoclaved.at I21°C, 15 lbs for 20 minutes and stored in room temperature.
It is another aspect of the present invention, wherein the total soluble solid (TSS) for pineapple juice is 17°Bx and for grape juiced is l6°Bx.

It is also aspect of the present invention, wherein the yeast and mould count (YMC) for pineapple juice is 5 CFU/ml and for grape juice it is 7 CFU/ml.
It is further aspect of the present invention, wherein the total plate count (TPC) for pineapple juice is 25 CFU/ml and for grape juice is 14 CFU/ml.
It is also an aspect of the present invention, wherein at 90 days pineapple juice sample had TPC of 14 CFU/ml and YMC is 4 CFU/ml.
It is yet another aspect of the present invention, wherein at 50 days grape juice sample had TPC of 10 CFU/ml and YMC is 1 CFU/ml.
BRIEF DESCRIPTION OF DRAWINGS:
For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:
FIG. 1 shows a TSS value of naturally preserved pineapple and grape juice. FIG. 1A shows a pH value of naturally preserved pineapple and grape juice. FIG. IB shows a Sensory analysis scoring of naturally preserved juice sample
DETAILED DESCRIPTION OF THE INVENTION:
The following detailed description illustrates by way of example and not by way of limitations.
Fresh,ripe, but firm and uniformly mature fruits were selected. Nutrient agar (Hi-media), Potato dextrose agar (Hi-media), sodium benzoate, citric acid, monosodium

glutamate, ascorbic acid, white sugar and phosphoric acid arc chemicals used and raw materials like Clove, Cinnamon, Ginger, Lemon, Honey, Pepper, Indian gooseberry, Indian borage, Pineapple fruit and Grape fruit are used.
Process: Grading:
The grading process is mostly done to remove diseased, damaged, or decayed fruits are rejected or trimmed. The fruits are graded according to their size and colour to obtain good quality of products.
Grapes are usually graded with hand and pineapple are graded after cutting into pieces or slices.
Washing:
Washing of the fruits is the most important and a necessary process. The washing process is done to remove the pesticide spray residue, dust from the fruits, and to remove spores (i.e. soil contains 1012 spores of microorganisms), so it is essential to wash the fruits (grape and pineapple) with water.
Peeling and cutting:
Pineapple was peeled using cutting knife, pineapple eye remover and the grape was
sorted for the further process.
The peeled pineapple fruits were cut into small pieces and was further processed.
Blanching;
The prepared pineapple and grape were blanched into a hot water (80 *C) for 3m in to remove the dirt. This process reduces number of microorganisms.
Cooling:

After blanching these fruits are dipped into cold water for 2min for belter handling and keeping them in good condition.
Juice extraction:
Generally juice is extracted from fresh fruits by crushing and pressing them. The pineapple and grape juice were extracted using a juice extractor.
Straining and filtration:
Fruit juices always contain varying amounts of suspended matter consisting of broken
fruit tissue, seed, skin, gums, pectin substances and protein in colloidal suspension.
Seeds and pieces of pulp and skin which adversely affect the quality of juice are removed by straining through a sieve or metal mesh. Removal of all suspended matter improves the appearance but often results in disappearance of fruity character and flavour. The juice extract was retained for the further process.
EXTRACTION OF CRUDE NATURAL PRESERVATIVES:
Preparation of lime extract:
Fresh lemon was bought from local market. The juice was squeezed using the
squeezer and the extract was used fresh as preservative.
Preparation of ginger extract:
Fresh ginger was brought from local market. It was grated and squeezed. Then the
extract was filtered and used fresh for preservative.
Preparation of Indian borage extract:
Their leaves were rinsed with water for 2 times. Then its leaves were separated and
was finely grinded using mortar and pestle. The finely grinded paste was filtered
using the filter paper. The plant extract was prepared fresh and used.
Preparation of Indian gooseberry extract:

Indian gooseberry was washed and cut into pieces. It was finely grinded using murlar and pestle. The extract was filtered using filter paper and the final extract was used fresh for the preservation.
Preparation of clove extract:
Clove extract was prepared by making the clove into fine powder and then weigh 0.25g of clove and add 25ml of distil water. Then allow it to boil at 8010 for lOmin. The aqueous solvent was filtered using the filter paper and the filtrate is used fresh.
Preparation of cinnamon extract:
Cinnamon extract was prepared by dissolving 0.5g of cinnamon powder in 5ml of distil water. Then allow it to boil at 80X2 for lOmin. Filter the aqueous solvent using the filter paper and use is used fresh for the preservation process.
PROCESSING AND PRESERVATION OF FRUIT JUICES USING NATURAL PRESERVATIVES:
PINEAPPLE JUICE:
The pineapple juice was centrifuged and the supernatant juice sample was alone filtered and used.
The TSS and pH of the filtered pineapple juice were observed and recorded.
The filtered pineapple juice were taken in a clean and sterile glass bottles (100ml). The juices were mixed along with the distil water in the dilution rate (70% fruit juice: 30% water).
After dilution the TSS and pH was checked. Then 7ml of preservative H was added and checked for TSS.
Then the various preservatives were added as tabled below:

After the addition of preservatives then, the juice sample were boiled @ 80 *C for 2min and it was bottled and then sealed using crown corking machine.
The bottled sample were autoclaved @121"C, 151bs for20min then it was stored and studied under the room temperature for the microbial growth analysis. The TSS and pH of the preserved juice sample were observed during the interval time of storage.
After the addition of preservatives then, the juice sample were boiled @ 8CC for 2min and it was bottled and then sealed using crown corking machine. The bottled sample were autoclaved @121 X2, 15lbs for 20min then it was stored and studied under the room temperature for the microbial growth analysis. The TSS and pH of the preserved juice sample were observed during the interval time of storage.
GRAPE JUICE:
The grapejuice was centrifuged and the supernatant juice sample was alone fdtered and used.
The TSS and pH of the filtered grapejuice were observed and recorded
The filtered grapejuice were taken in a clean and sterile glass bottles (100ml). The juices were mixed along with the distil water in the dilution rate (60% fruit juice: 30% water).
After dilution the TSS and pH was checked. Then 5ml of preservative H was added and checked for TSS.
Then the various preservatives were added as tabled below:
After the addition of preservatives then, the juice sample were boiled @ 80*0 for 2 min and it was bottled and then sealed using crown corking machine.

The bottled sample were autocJaved @I2I TD, I5lbs for20min then it was stored and studied under the room temperature for microbial growth analysis.
The TSS and pH of the preserved juice sample were observed during the interval time of storage.
QUALITATIVE PHYTOCHEMICAL ANALYSIS:
The fruit juice samples is tested for the presence of bioactive compounds by using
following standard methods.
TEST FOR CARBOHYDRATES
IODINE TEST
Crude juice was mixed with 2ml of iodine solution.
A dark blue or purple coloration indicated the presence of phenols and tannins.
TEST FOR FLA VONOIDS
ALKALINE REAGENT TEST
Crude juice was mixed with 2ml of 2% solution of NaOH.
An intense yellow colour was formed which turned colourless on addition of few drops of diluted acid which indicated the presence of flavonoids.
TEST FOR SAPONINS
Crude juice was mixed with 5ml of distilled water in attest tube and it was shaken
vigorously.
The formation of stable foam was taken as an indication for the presence of saponins.
TEST FOR GLYCOSIDE
SALKOWSKI's TEST
Crude juice was mixed with 2ml of chloroform.
Then 2ml of concentrated H2S04 was added carefully and shaken gently.
A reddish brown colour indicated the presence of steroidal ring, i.e., glycone portion of the glycoside.

TEST FOR PHENOLS AND TANNINS:
Crude juice was mixed with 2ml of 2% solution of Fecl3.
Blue-green or black coloration indicated the presence of phenols and tannins.
TEST FOR REDOING SUGAR
Juice was shaken was mixed with distilled water and filtered.
Filtrate was boiled with Fehling's solution A&B for lOmin. Red precipitates indicates the presence of reducing sugar.
TEST FOR STERIODS:
Crude juice was mixed with 2ml of chloroform and concentrated H2S04 was added
sidewise.
A red colour produced in the lower chloroform layer indicated the presence of steroids.
Another test was performed by mixing crude extract with 2ml of chloroform.
Then 2ml of each of cone. H2S04 and acetic acid were poured into the mixture.
PROCESSING AND PRESERVATION FRUIT JUICES USING CHEMICAL PRESERVATIVE AND NATURAL PRESERVATIVE:
Pineapple juice and grape juice were centrifuged, autoclaved and the chemical preservative such as sodium benzoate, citric acid, ascorbic acid and sodium glutamate were added. Then the preserved fruit juice were stored under room temperature. 0.3 % of preservatives were added.
Natural preservative method was also processed and stored under room temperature. Were the chemical preservative fruit juice sample was autoclaved first and then preservatives were added once the temperature was bought down. The natural preservative fruit juice was autoclaved after the addition of natural preservatives.
TOTAL SOLUBLE SOLIDS (TSS) ANDpHANALYSIS:

TSS- Natural preservative process
TSS of both pineapple juice and grape juice was observed using refractometer and
recorded. At the initial stage the TSS of naturally preserved pineapple juice had 17° Bx and grape juice had l6°Bx. After the 20 days of storage period the TSS of juice samples was reduced by 2% and later it was maintained with the same value of 14° Bx.
pH — Natural preservative process
At the initial stage the pH of pineapple juice was 4 and the pH was maintained the same till 50 days of storage and there was a change in pH at 90 days sample (Fig. 5.5). The decrease in the pH value indicates that the juice has become acidic which results as the* process of fermentation as more acidic is been produced. Then the pH for grape juice at initial was 4 and it maintained the same pH till 50 days of storage.

QUALITATIVE PHYTOCHEMICAL ANALYSIS RESULTS:
Qualitative phytochemical screening was performed for both pineapple juice and grape
juice samples (Fig. 5.8). The screening result showed that both the sample was abundant in phytochemicals such as vitamins, carbohydrates, glucose and reducing sugars. The sample does not consist of flavonoids, saponin, glycosides and steroids.
Microbial analysis of natural preservatives
The quality of microbial analysis plays the main and important role in the preservation
process. Pineapple juice and grape juice samples of 0 day, 20 day, 50 day and 90 day) was analysed for microbial growth. TPC and YMC was performed by pour plate technique.

YMC FOR PINEAPPLE.JUICE & GRAPE JUICE:
The yeast and mold count was performed by pour plate method with 0.1ml of sample in
PDA media and incubated @ 32 "C for 48hrs for the growth observation. After the
incubation period the YMC was counted and recorded for both pineapple juice and grape
juice sample.
The yeast and mold count (YMC) for pineapple juice is 5 CFU/ml and for grape juice it is 7 CFU/ml
TPC COUNT FOR PINEAPPLE JUICE & GRAPE JUICE:
The total plate count was performed for by pour plate technique with 0.1ml of diluted
samples (9ml dis. H20+lml of sample). And was incubated @ 32°C for 24 hrs. After the
incubation period the TPC was observed and recorded for pineapple juice and grape
juice.
The total plate count for pineapple juice is 25 CFU/ml.

The total plate count for grape juice is 14 CFU/ml.
As the microbial growth reduces it shows the effectiveness of the preservative that reacts with the juice samples.
Sensory analysis of naturally preserved juice sample
The juice samples were tasted and all the characterization and attributes were recorded.
The samples had good taste and aroma. The juice samples were not spoiled over 90 days of storage under room temperature. The sensory analysis was performed for both pineapple juice and grape juice sample and their taste characterization and attributes were recorded.

Natural preservative juice sample
Both the pineapple juice and grape juice sample was preserved with natural preservative
and was thermally processed. The processed fruit juice sample was stored for 90 days for shelf life study of natural preservatives .in fruit juice sample. The preserved fruit juice samples maintained good characteristic and attributes. The juice samples were fresh and retained its flavor over the storage period. The pineapple juice had no contamination over

90 days of preservation and grape juice also had no contamination over 50 days of
storage period. The natural preservative has more effect in both grape and pineapple juice
samples.