FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
PROVISIONAL SPECIFICATION [See section 10]
PRESERVATIVES FROM CHITIN
DERIVATIVES;
CAMLIN FINE CHEMICALS LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS CAMLIN FINE HOUSE, PLOT NO. F/ll & F/12, WICEL, MIDC, MAROL, CENTRAL ROAD, ANDHERI (EAST), MUMBAI - 400 093, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION
DESCRIBES THE INVENTION.

TECHNICAL FIELD
The invention relates to compositions, process for production of compositions of chitin and chitosan that act as a respiration controller, anti-microbial, microbiostatic, efficient remover of microbes and anti-oxidative for keeping freshness of fruits, vegetables, cut flowers, meat, fish and other processed food products and methods of applying the same and the applicators for the same.
BACKGROUND
Chitin and chitosan have been used in the past as post harvest preservatives of fresh fruits and vegetables. The chitosan used is isolated from prawn shell and treated for varying degree of acetylation and dissolution in 1 - 2 % acetic acid.
Chitosan has been used for improving the shelf life of various fruits and vegetables such as citrus [Magnetic Resonance Imaging 22 (2004) 27 - 37], carrot [Food Control 17 (2006) 336 - 41], lettuce and strawberry [Food Microbiology 21(2004)703-14], and litchi [Postharvest Biology and Technology 38 (2005) 128 - 36] wherein the chitosan used is of more than 43 Kd ( Kilo Dalton) size and 93% de-acetylation. Anti-oxidant property of chitosan has been studied on Salmon [Food Chem 101 (2007) 308-11] where chitosan of three different molecular weights has been used. Solution (1%) of 30 kD chitosan showed results equivalent to chemical anti-oxidant BHT, and were superior to those obtained using chitosan of 90 kD and 120 kD molecular weight. Useful preservation activity of chitosan obtained from prawn shell has been reported for various foods such as eggs, meat, sausage, sea food, tofu, Juices, Mayonnaise, Kimchi, Noodles, Rice cake, soy bean sprouts, starch jelly etc. In case of milk preservation chitosan of molecular weight varying from 0.2 - 30 kD has been described [Korean J. Food Sci. Tech. 32 (2000) 806 - 13]. In case of bread three different molecular weights of chitosan - 2 kD [J Chitin Chitosan 7 (2002) 214 - 8], 120 kD [Korean J Food

""Nutrition 11 (2003) 309 - 15] and 493 kD [J Chitin Chitosan 7 (2002) 208 - 13] have been used successfully for extending its shelf life, sustaining weight, and retarding starch degradation. Antimicrobial activity of chitosan has been reported against various bacteria yeasts and molds such as Aeromonas, Bacillus, Bifidobacterium, brochothrix, Clostridium, Enterococcus, Escerechia, Lactobacillus, Leuconostoc, Listeria, Micrococcus, Pediococcus, Photobacterium, Pseudomonas, Salmonella, Shigella, Staphylococcus, Vibrio, Candida, Cryptococcus, Saccharomyces, Schizosaccaromyces, Zygosaccharomyces, Aspergillus, Botrytis, Cladosporium, Penicillium, Rhizopus, etc. and has been reviewed [J Food Sci 72 (2007) R 87 - R100]. Except for the few examples mentioned specifically, all prior art methods have used the high molecular weight chitosan obtained from prawn shell with varying and undefined molecular weights and degree of deacetylation.
US patent no. 5,374,627 describes method of control of plant diseases and damage by certain pests in agricultural plants by compositions containing 1 part by weight of a chitosan hydrolyzate with an average molecular weight of 10,000 to 50,000, obtained by acid hydrolysis or enzymatic hydrolysis of chitosan.
The patent application WO 03070008 discloses antimicrobial compounds and their methods of application wherein the product for use is a liquid obtained by using organic acids of 3 -30 carbons and the claimed product is useful in the preservation of the acidic foods only. In one of the example chitosanase enzyme is used for degradation of chitosan.
Although various chemical modifications of chitosan are known in the literature that lead to solubilization of chitosan at neutral to alkaline pH, the applications of chitosan in food are only using solution of chitosan in organic acid preferably acetic acid. The solution thus obtained is of low pH (2.0 - 4.0) depending on the concentration of the acid used. The acid used in the said formulation adds taste to the product to be treated, and/or adversely affects the shelf life. In case of banana, acetic acid is known to develop senescent spots [black

discolouration of the skin] which is undesired. Acids used in the chitosan formulation are responsible for curdling of the milk and milk products. The net positive charge developed on the chitosan molecule because of the low pH is responsible for separation of anionic components from the product to be treated [such as inorganic salts, fatty acids], which adsorb on the positively charged chitosan. All these reactions are undesired and can be avoided by making the formulation as described in the present invention.
A class of water soluble chitosan as described in the literature is of complexes of chitosan with respective organic acids which are typically described as chitosan acetate, chitosan lactate etc. These are dry powders of chitosan acid complex, derived by simple mixing followed by removal of un-reacted acid and drying. Such complexes are of poor chemical definition, rather unstable, and still have the problems associated with low pH like that of the chitosan solution.
The partial degradation products of chitin and chitosan like chito-oligosaccharides are known to be water soluble. However, these molecules have varying degree of biological activities such as respiration control, microbio-stasis, coating characteristics etc. These are generally very weak in the desired levels of activities as compared with the products developed using the method(s) claimed in the present invention.
This invention embodies novel compositions of chitin/ chitosan for treatment as anti-respiratory, anti-microbial and anti-oxidative for keeping freshness of natural products, processes for production of the said novel compositions, novel methods for the said treatment to the said natural products for enhancing or improving their shelf life or storage stability. This invention further embodies a novel class of proteolytic microbial enzymes that have chitinase action too and their use in developing novel chitosan compositions of this invention.

The invention also embodies methods of keeping freshness of natural products for longer time. The invention further embodies methods of application of the said compositions and equipment useful for the purpose of applying the said compositions to the natural products. fruits, vegetables, meat, fish and other food products. Illustrative list of natural products covered by the scope of this invention includes food products and non-food natural living or non-living products, further including without limitation, fruits, vegetables, meat, fish, cut flowers, milk and food products in general.
In one of the embodiment of this invention chitin/chitosan are dissolved in organic acid and the resultant solution shows anti-microbial activity. The concentration and volume of the organic acid are crucial in exhibiting the activity.
In yet another embodiment of this invention chitin/chitosan hydrolyzate are obtained by using purified enzymes or crude form of enzymes or active form of microbial cell extract. The said hydrolysate shows improved anti-microbial activity upto a certain limit of decrease in average molecular weight, and within specific range of degree of substitution of the free amino group by either acetyl or any other anionic group. In a further embodiment of the invention, enzymatically hydrolyzed and de-acetylated chitin/chitosan show better antimicrobial activity than chemically derived product.
In a further embodiment of this invention a class of electrophoretically homogeneous enzymes isolated from microorganisms has been found to have a proteolytic activity as well as chitinase activity, which may be designated as proteo-chitinases. In an embodiment of this invention, the said proteo-chitinases are used to hydrolyse chitin / chitosan in a process to make products of this invention. Although proteo-chitinases of this invention are isolated from microbes, this class of enzyme, derived from whichever source shall perform the same function of hydrolysis of chitin or chitosan and this invention extends to their use also for the purpose of chitin / chitosan hydroysis and further for developing products of this invention.

In another embodiment of this invention the hydrolysate obtained is further modified, i.e. de-acelyated using xylan acyl transferses and this deaceylated form of hydrolysate shows further improvement in anti-microbial, anti-oxidant property and acts as an anti-respiration agent too. The hydrolysate having molecular weight in lower range of 8 - 100 kD are more effective than higher and / or lower molecular weight hydrolysate. Preferred method of getting xylan acyl transferses in this invention is from microorganisms. These enzymes, isolated as crude or purified extract from any other source can be predicted to have the same deacylating properties and yield the same products as are prepared in the preferred embodiments of this invention from microorganisms.
In yet another embodiment of this invention Chitosan succinate sugar derivative is prepared showing variable solubility in water and also shows varying anti-microbial property. Sugar may be cane sugar or any other sugar.
In another embodiment of this invention Chitin or chitosan includes derivatives prepared from an inorganic acid. This is exemplified by using phosphorus acid. But any other inorganic acid may also be used in its place.
Chitosan has positive charge, and any acid whether organic or inorganic will form coordinate bond with the charged amino group on chitosan. These complexes have been named as respective salts of chitosan e.g. chitosan hydrochloride (HC1), chitosan acetate (acetic acid) etc. These salts dissolve in water, and the pH of the solution is generally acidic. The salts also do not posses anti-microbial activity. When we try to increase the pH of these solutions by addition of any alkali - typically NaOH, the chitosan precipitates before the pH has become neutral. This is because the salt is not stable at the said pH. In our processes we have two

xypes of modifications which are unique: 1. Formaldehyde mediated reaction and anhydride mediated reaction.
Both these reaction products are generally stable at akaline pH. Now the solubility of the product at alkaline pH is a function of net charges on the molecule. These can be added within either of the said 2 reactions, or by simple ionic interaction (salt formation) with any acid. Typically inorganic acids are preferred as the salts are more stable. 2. The soluble chitosan derivatives developed by various methods described herein have partial substitution and retain both positive and negative charges. Thus different charged molecules are attached to chitosan molecule, thereby rendering it useful as a carrier molecule for active groups, such as antimicrobial groups, or any other active group that have some functional purpose. The result is that the efficacy of the functional group that is attached to such molecule improves at a very small concentration. Herein, water soluble chitosan compositions prepared by the methods of invention have been seen to be effective carriers of n acetyl cysteine and Copper Sulphate residues, the later has been demonstrated in Example 19. Same performance is expected to occur for any other functional group whose reside can be as effective as when it is not carried by water soluble chitosan molecule. Water soluble chitosan molecule used here is prepared by methods of this invention. However, water soluble chitosan molecule prepared by any other existing or inventive method shall be equivalent to this molecule and shall be considered as being covered within the scope of this invention unless there is any reason for which it can not carry any other active for any reason.
A yet another embodiment of this invention discloses a method of enhancing storage stability of natural products by treating with chitin derivatives. One embodiment of this aspect of invention discloses a method of enhancing storage stability of natural products by treating with chitin derivatives that retard life activities that are responsible for reducing storage stability or lead to ageing by slowing down or arresting such activities. Illustrative list of the

said life activities include, without limitation, respiration, oxidation, ethylene formation, transpiration, as well as arresting aging of the fresh fruits, vegetables, meat, fish, processed and unprocessed food preparations and like. A person of ordinary skill in the art will be able to extend this list to many other life activities that reduce storage stability. The said coating can be full or partial and can be obtained by use of a method of application. The said method of application includes, without limitations, spraying, dipping, micro-spraying or by use of nano-particles, indirect fogging and like. In case of processed food such as bread, noodles etc. the said formulation can be added in the dough, and / or applied to the surface of the processed food by one of the various methods listed above.
In the following are described experiments conducted that serve as non limiting illustrations of how the invention is performed. Any modifications or variations in the parameters, including but not limited to process for producing hydrolysates and further modifications, the method of use of the products and their various steps used are merely illustrative and any equivalents of them that are obvious to a person skilled in the art and capable of achieving the same objective may be used in their place and yet they shall be considered as included in the scope of this specification.
Figures and their description:
Figure 1: Photograph of the untreated (left) and treated (right) banana on 12th day of the storage.
Figure 2: The MRI scan of the representative tomatoes.
Figure 3: A photograph of difference in the appearance of treated and untreated cut apple on day 4.
Figure 4; The untreated cut apple developed fungal growth visible as black patches in the photograph.

Example 1: PRODUCTION OF WATER SOLUBLE AND INSOLUBLE CHITOSAN LACTATE
Chitosan [70 DDA, 100 mesh] 5 gm is taken in a round bottom flask and 20 ml distilled water and 80 ml methanol (99%) is added to it. Any other grade of chitosan can be used here. The suspension is mixed well by stirring at 100 rpm for 30 min. Lactic acid is added in different concentrations to this mixture and stirring is continued for 2 hours. The temperature is maintained below 30 degree C and preferably at 25 degree C. The suspension is filtered and the solid is air dried. The free flowing powder and lumps (if any) are separated by sieving. The free flowing powder is then further dried in hot air oven [70 degree C for 3 - 6 hours]. Lumps are elastic particles that do not break to free flowing particles of 100 mesh or smaller. Solubility of the free flowing particles is checked by adding 100 mg powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The effects of different concentrations of lactic acid are given in the Table 1.
Table!: Effect of different quantities of lactic acid used with 5 gm chitosan.

Lactic acid (ml) Formation of lumps Solubility in water
6.5 +++ Dissolves completely
5.0 ++ Dissolves completely
3.5 + Dissolves completely
2.0 - Dissolves completely
1.0 - Forms lumps that dissolve after 30 min
0.5 " Forms lumps that do not dissolve up to 60 min.
+++ indicates 60% or more lump formation. ++ 35% Jump formation
+ about 15% lump formation and lump size is small _ no lump formation

Example 2:
ADDITION OF SUCCINATE GROUP TO CHITOSAN
Chitosan [70 DDA, 100 mesh] 5 gm is taken in a round bottom flask and 20 ml distilled water and 80 ml methanol (99%) is added to it. Any other grade of chitosan can be used here. The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 70 degree C. Succinic anhydride is added in different proportions ranging from 15 g to 0.1 gm to this mixture and stirring is continued for 1 hour. The temperature is maintained between 60 -70 degree C and preferably at 65 degree C. The suspension is allowed to cool and filtered and the solid is air dried. The free flowing powder is formed by breaking the loose lumps mechanically and sieved using 100 mesh. No lumping was observed. The free flowing powder is then further dried in hot air oven at 70 degree C for 3 - 6 hours. Solubility of the free flowing particles is checked by adding 100 mg powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is soluble in water when more than 0.5 gm succinic acid is added to 5 gm of chitosan in the said reaction.
Example 3:
SIMULTANEOUS ADDITION FO SUCCINATE AND LACTATE TO CHOTISAN
The reaction is carried out exactly as described in example 2 wherein the chitosan is replaced by the reaction product obtained in a reaction described in example 1 where 1.0 gm or 0.5 gm lactic acid is used. Further 0.5 gm of succinic anhydride was used for 5 gm of the powder. Coarse granular material was formed which was soluble in water when tested as described in example 1,

Example ASEQUENTIAL ADDITION OF SUCCINATE AND LACTATE TO CHITOSAN
The reaction was carried out as per example 2 wherein 5 gm chitosan is reacted with 0.5 gm succinic anhydride. After the reaction was complete and the reaction mixture was cooled to 30 degree C and lactic acid - 0.5 or 1.0 ml was added. The mixture was stirred for 1 hour, filtered and processed further as described in example 1. There was no lump formation. The dry powder was completely soluble in water within 5 min under the described test conditions.
ExampleS:
IDENTIFICATION OF ENZYME HAVING PRIMARY PROTEINASE ACTIVITY AND CHITINASE CROSS REACTIVITY
Crude microbial culture filtrate was concentrated by ammonium sulphate precipitation dissolved in 0.1% SDS. The protein fractions were separated by denaturing SDS PAGE electrophoresis. The protein fractions were separated on these gels as homogeneous independent bands. These bands were characterized for their activity by three different methods; coumassie blue staining for protein detection, casein clearance assay for proteinase activity and Ramezol Brilliant Blue Chitin clearance assay for chitinase activity. An electrophoretically homogeneous band was detected that showed proteinase as well as chitinase activity — Proteo-chitinase. This band was used for developing hydrolysate of chitin for developing product of this invention.
Example 6:
PRODUCITON OF PROTEASE CAPABLE OF CLEAVING CHITIN AND CHITOSAN
Microbial culture(s) were identified that are capable of production of acid or neutral protease(s) and endo-chitinase. The enzyme fraction isolated from the respective microbial cultures that had proteinase as well as chitinase activity (proteo-chitinase) was incubated with

1% chitosan solution in 1% acetic acid. The protease degraded the chitosan partially resulting in reduction in the viscosity of the substrate. The cultures identified to posses promising proteinase as well as endo-chitinase reaction (Proteo-chitinases) were grown on a large scale for enzyme production. Specifically selected were Aspergillus niger and Bacillus sp. which were grown under shake flask conditions in casein yeast extract medium. The culture filtrate was characterized for enzyme activity, and the crude proteo-chitinase enzyme was recovered by ammonium sulfate precipitation [90% saturation]. The enzyme activity of the ammonium sulfate precipitate was determined by protein determination by colorimetric method, and known quantity of proteo-chitinase enzyme was added to chitosan for its modification.
Example 7:
PRODUCTION OF XYLAN ACYL TRANSFERASE CAPABLE OF DEACETYLATING CHITIN AND CHYTOSAN
Microbial culture(s) were identified and isolated that are capable of production of xylan acyl transferase. The deacetylation of chitosan under the test conditions was done by these enzyme(s). The reaction mixture showed solubilization of chitin as viscosity of the solution increases. The occurrence of free acetyl group is detected by TLC. The cultures identified to possess chitin de-acetylation property were grown on large scale for enzyme production. Cultures selected were of Aspergillus niger and Bacillus sp. and were grown under shake flask conditions in wheat bran yeast extract medium. The culture filtrate was characterized for enzyme activity, and the crude enzyme was recovered by ammonium sulfate precipitation [90% saturation]. The enzyme activity of the ammonium sulfate precipitate was determined, and known quantity of enzyme was added to chitosan for its modification.

Example 8:
ENZYMATIC HYDROLYSIS OF CHITOSAN
Chitosan 5 gm was added to methanol 70 ml and distilled water 30 ml. This mixture was stirred continuously at 100 ppm. Enzyme capable of cleaving chitin [described in example 5] was added at 10 - 1000 I.U. per gm chitosan concentration. The reaction was carried out at temperatures ranging from 25 - 95 degree C for 30 min to 5 hours. The preferred conditions were 100 I.U. enzyme per gm chitosan, temp, of 50 degree C and an incubation period of 2 hours. The reaction product was recovered by filtration and drying as described in example 2. The unmodified chitosan [1 gm] when dissolved in 100 ml distilled water acidified using 2 ml acetic acid solution, showed viscosity of 80 - 100 cps. The enzymatically modified chitosan [1 gm] when dissolved in 100 ml distilled water acidified using 2 ml acetic acid solution, showed viscosity of 20 - 30 cps.
Example 9:
ENZYMATIC DEACETYLATON OF CHITIN
Chitin 5 gm was added to 100 ml distilled water. This mixture was stirred continuously at 100 ppm. Enzyme capable of de-acetylation of chitin [example 6] was added at 500 - 1000 I.U. per gm chitin concentration. The reaction was carried out at temperatures ranging from 25 - 95 degree C for 8 to 24 hours. The preferred conditions were 800 I.U. enzyme per gm chitin, temp, of 50 degree C and an incubation period of 8 hours. The reaction product was recovered by filtration and drying as described in example 2. The unmodified chitin was completely insoluble in water. The enzymatically modified chitin [10 gm] when stirred in 100 ml distilled water acidified using 2 ml acetic acid solution, showed viscosity of 30 - 50 cps, indicating partial solubilization of chitin as a result of de-acetylation.

Example 10:
THE POST HARVEST PRESERVATIVE CHITIN / CHITOSAN COMPOSITION AND PREFERRED PROCESS OF MANUFACTURING OF THE SAME
Chitosan [50 - 70 DDA, 100 mesh] 5 gm is taken in a round bottom flask and 20 ml distilled water and 80 ml methanol (99%) is added to it. Any other grade of chitosan can be used here. The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 70 degree C. Succinic anhydride is added in different proportions ranging from 15 g to 0.1 gm to this mixture and stirring is continued for 1 hour. The temperature is maintained between 60 -70 degree C and preferably at 65 degree C. The suspension is allowed to cool. Lactic acid 0.5 ml, and enzyme for deaceylation 4000 I.U was added and the mixture was stirred for 7 hours. Chitinolytic enzyme 500 I.U. described in example 5 was added and the stirring was continued for 1 hour. The suspension is allowed to cool and filtered and the solid is air dried. The free flowing powder is formed by breaking the loose lumps mechanically and sieved using 100 mesh. No lumping was observed. The free flowing powder is then further dried in hot air oven at 70 degree C for 3 - 6 hours. Solubility of the free flowing particles is checked by adding 100 mg powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The powder is soluble in water when more than 0.5 gm succinic acid is added to 5 gm of chitosan in the said reaction.
Example 11:
PRODUCTION OF WATER SOLUBLE AND INSOLUBLE CHITOSAN SUCCINATE -SUGAR DERIVATIVE
5 gm of Chitosan succinate derivative as described in example 10, is taken in a round bottom flask and 100 ml methanol (99%) is added to it. The suspension is mixed well by stirring at

100 rpm for 30 min. and heated to 60 degree C. The temperature was maintained through out the reaction. Sucrose - in the range of 1 gm to 0.02 gm (see Table No. 2) was dissolved in 10 ml water, and same was added to the suspension. Mixture was stirred for 15 min. Formaldehyde 5 ml was added to this mixture drop-wise over 30 min. The reaction was continued at 60 degree C with constant stirring further for 1 to 6 hours. This is termed as reaction time in the Table No. 2. The reaction mixture was allowed to cool down to room temperature. Subsequently the product was recovered by filtration. The solids were washed with 100 ml 90% methanol and air dried. The product was characterized as free flowing powder or lumps. The solubility of 0.5 % powder was determined in distilled water (pH 6.5) and 1% acetic acid. The results of these experiments are summarized below:
Table. No. 2.

Sucrose in gm Reaction time in Hours Product form Solubility in water Solubility in 1% acetic acid
1 6 Powder Insoluble Insoluble
0.5 6 Powder Insoluble Insoluble
0.1 6 Lumps/Gel Soluble Insoluble
0.02 6 Lumps/Gel Soluble Insoluble
0.02 3 Lumps/Gel Soluble Soluble
0.02 1 Powder Soluble Soluble
All the water soluble products had varying degree of anti-microbial activity, which was consistently better than that of anti-microbial activity of the starting material (product of example 10)
The anti-microbial activity of these products was tested using E. coli, and the protocol described in example 14. The results of the test are as follows:

Table : initial count of E coli was 4X 10*7

Product/ concentration 1% 0.5% 0.1% 0.033%
Product of example 10 2X10*3 2X10*3 4X10*3 1X10*4
Product 10 with sucrose 0.1 gm 7X10*2 4X10*2 4X10*2 1X10*3
Product 10 with sucrose 0.02 gm 6h 5X10*2 2X10*2 4X10*2 90
Product 10 with sucrose 0.02 era lb. 2X10*2 2X10*2 4X10*2 109
The reactions were carried out as described above but replacing sucrose with glucose or lactose 0.02 gm each for 3 hours. The products of these reactions were soluble in water and showed 100% anti-microbial activity against E coli when by the method described above.
Example 12:
CHITIN / CHITOSAN COMPOSITION MADE BY USING PHOSPHORUS ACID AND CARRYING FORMALDEHYDE RESIDUES
Chitosan [50 - 70 DDA, 100 mesh] 5 gm is taken in a round bottom flask and 100 ml methanol (99%) is added to it. Any other grade of chitosan can be used here. The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 60 degree C. Phosphorus acid (H3PO3) in different proportions ranging from 0.5 g to 15 gm is dissolved in 20 ml of water and added drop-wise to this mixture by stirring for 1 hour. Formaldehyde 5 ml is then added in drop-wise manner. The temperature is maintained between 60 - 65 degree C and preferably at 63 degree C for 2 - 6 hours after the addition is complete. The suspension is allowed to cool and filtered and the solid is air dried. The free flowing powder is formed by breaking the loose lumps mechanically and sieved using 100 mesh. No lumping was observed. The free flowing powder is then further dried in hot air oven at 70 degree C for 3 - 6 hours. Solubility of the free flowing particles is checked by adding 100 mg powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is checked visibly. The solubility is also checked at pH 8, 9, and 10 adjusted using dilute NaOH, or Tris buffer.

The products formed using chitosan and phosphorus acid in 1:1 and 1:2 ratio showed solubility in the broad range of pH (7 -10), forming slightly turbid to clear solutions. These solutions showed varying degree of anti-microbial activity. The antimicrobial studies were conducted using Pseudomonas aeruginosa NCIM 2200 (ATCC 9027) capable of growing at a broad pH range. The overnight culture grown in the nutrient broth was inoculated (100 micro L) again in nutrient broth (10 ml) of varying pH (see the table) without any addition (control) and with chitin-phosphorus acid product. The cultures were incubated on shaker at 25 degree C for 8 h, and the growth was recorded as optical density (OD) of the broth at 600 nm. Low optical density (than control) indicates less growth, and some anti-microbial activity. No growth - zero OD - means 100% inhibition of microbes.

C:P1:1 C:P1:2
pH7 C 1.74
0.01% 1.56 1.53
0.05% 0 0
0.10% 0 0
pH8 C 1.73
0.01% 1.39 0
0.05% 0 0
0.10% 0 0
pH9 C 1.69
0.01% 1.39 1.68
0.05% 0 0
0.10% 0 0
Example 13:
APPLICATION OF THE PRESERVATIVE COMPOSITION
Solutions were prepared from 10 gram of chitosan and its product described in example 1, 2, 4 and 10 by dissolving in 2% acetic acid and water respectively to make a volume of 1000 ml solution in a vessel of 15 L capacity. 3 kg tomato or 2 kg banana were dipped in the solution for 15 sec. When the material was taken out from the container, excess solution was allowed

to drain in the same container for 1 min. This dipping action was repeated with next five batches of fresh fruit. Thus a total of 15 kg tomato or 10 kg banana was dipped in 10 L solution. Then the remaining solution in the vessel was accurately measured and loss of liquid was recorded as difference in the initial and final volume. This was divided by the total amount of fruit dipped in it, giving the consumption of solution per kg of the fruit dipped in. The results of this experiment are given in table 2.
Table 2:

Product Consumption in Kg tomato ml per Consumption in ml per kg banana
Chitosan 12 19
Chitosan: Lactic acid 1:0.4 as per example 1 9 10
Chitosan: Succinic anhydride 1:0.1 per example 2 8 8
Product of example 4 3 5
Product of example 10 3 3
Example 14:
ANTIMICROBIAL ACTIVITY OF CHITOSAN COMPOSITIONS
Chitosan and its products described above were dissolved in 2% acetic acid and water respectively. 300 mg of the product was used to make 10 ml solution. E.coli and Bacillus subtilis cultures were grown overnight (10*9 cells/ml density) diluted 100 folds with sterile liquid broth and used for antimicrobial assay. The assay was set by mixing diluted cultures with stock solution of chitosan or product as follows:

Culture vol. In ml 7 8,5 9.7 9.9
Chitosan/ product vol. in ml 3 1.5 0.3 0.1
Concentration of product (%) 1 0.5 0.1 0.033
The mixture was incubated at 37 degree C for 20 min. The total viable count of the mixture was determined. This when compared with the initial count of the culture gives us the

'microbicidal effectiveness of the product. The data for E coli and Bacillus subtilis is given in table 3 and 4. Table 3: initial count of E coli was 4X 10*7

Product/ concentration 1% 0.5% 0.1% 0.033%
Chitosan 2X 10*7 4X 10*7 4X 10*7 4X 10*7
Chitosan: Lactic acid 1:0.4 6X 10*5 1X10*6 2X 10*7 2X 10*7
Chitosan: Succinic anhydride 1:0.1 8X 10*6 2X 10*7 2X 10*7 2X 10*7
Product of example 4 9X10*5 2X 10*6 9X 10*6 3X 10*7
Product of example 10 2X10*3 2X10*3 4X10*3 1X10*4
Table 4: initial count of Bacillus subtilis 3X10*7

Product/ concentration 1% 0.5% 0.1% 0.033%
Chitosan 3X10*7 3X 10*7 4X 10*7 4X 10*7
Chitosan: Lactic acid 1:0.4 4X 10*5 3X 10*6 1X10*7 7X 10*6
Chitosan: Succinic anhydride 1:0.1 3X 10*6 2X10*6 8X 10*7 6X 10*7
Product of example 4 7X 10*5 1X10*6 5X 10*6 1X10*6
Product of example 10 1X10*4 2X10*4 4X10*5 1X10*5
The mixture (0.1%) was incubated in refrigerator (at temperature 4-10 degree C) up to 7 weeks. Increase in the turbidity was visually detected and scored as +. No increase in the turbidity (-) indicates microbiostatic activity.
Table 5:

Product/ microbial culture Weeks E coli B subtilis

3 5 7 3 5 7
Chitosan + + + + + +
Chitosan: Lactic acid 1:0.4 - + + - - +
Chitosan: Succinic anhydride 1:0.1 + + + - + +
Product of example 4 - - + - - +
Product of example 9 +
Example 15:
EFFECT OF PRESERVATIVE COMPOSITIONS ON STORAGE SABILITY
When chitosan solution, chitosan processed by example . 6 and 7 were used to coat banana and strawberry fruits - 100 fruits for each set, the lowest number of microbial spoilages ( 22

and y for the respective fruits) were observed after 5 days of storage at room temperature and 25 for chitosan processed by example 6 method-Example 16: IMPROVEMENT IN THE SHELF LIFE
Freshly harvested (<30 h of harvesting) green banana are treated using 0.1% solution of the product from example 10 in water. The treatment can be by dipping of banana in the said solution for nominally 10-15 sec, or by direct spray of the solution on the banana with a suitable device so as to make the surface of banana wait, or by passing the banana through a closed chamber which is saturated with the fog of the said solution (indirect fogging). The treated banana are air dried. These banana are stored at room temperature (20 - 32 degree C) and a relative humidity of 40 - 60%. The banana from the same lot which are identical to the treated banana are kept as 'untreated control'. Both treated and untreated banana are stored under identical conditions for up to 15 days. The treated banana maintained green color, and weight where as the untreated banana lost weight and developed discoloration. The photograph of treated and untreated banana taken on 12th day, shows the visual differences. The treated banana had better physiological condition as tested chemically by determination of their total solids (BRIX contents), reducing sugars, and weight loss. The data is shown in table 5 and figure 1.

"Table 5: Results of the quantitative tests conducted on treated and untreated control banana on 6l day of the storage.

Parameter Treated Control
Weight loss 2.6 8.7
BRIX 22-24 20-22
RS 800-1100 300 - 850
Photograph of the untreated (left) and treated (right) banana on 12vh day of the storage is shown in Figure 1.
Further to the storage for 8 - 15 days, both the treated and untreated control banana were treated with ethylene. The treated banana ripened normally, whereas the untreated control banana either became hard and did not ripen, ox turned black and soft. Both the types of banana from the untreated lot were not acceptable for consumption.
Example 17:
POSITIVE PHYSIOLOGICAL CHANGES AND MICROBIO-STATIC ACTIVITY:
The whole tomato and cut apple were treated with the product from example 10, exactly as described in example 14 for banana. The treated tomato retained its weight, texture and freshness for 30 days as against untreated control tomato, which wrinkled in 12 - 15 days resulting in weight loss. Physiological changes in the treated and control tomatoes were monitored on 7th day of storage using MRI scanning technique. The fleshy skin of the treted tomato was more uniform in its color density. The fleshy skin of the untreated control tomato has dark patches that are indicative of uneven and more drying.

"" The MRI scan of the representative tomatoes is included in the Figure 2.
The pieces of cut apple were packed in polythene bag or transparent plastic container and stored in refrigerator at temperature of 4 - 9 degree C. The cut apple from control started becoming brown from day 2 and turned soft by Day 3, where as the treated cut apple retained its color, taste, and hard texture for 6 days. The photograph (Figure 3) shows the difference in the appearance of treated and untreated cut apple on day 4.
When treated and untreated cut apple pieces were packed in transparent plastic container and stored as describe above for up to 25 days. The untreated cut apple developed fungal growth [black patches in the photograph in Figure 4], whereas the treated cut apple did not develop any fungal or other microbial growth. The results are depicted in the following photograph.
EXAMPLE 18: CHITIN / CHITOSAN COMPOSITION USING PHOSPHORUS ACID AND HYDROCHLORIC ACID
Chitosan [50 - 70 DDA, 100 mesh] 750 gm is taken in a round bottom flask and 15000 ml methanol (99%) is added to it. Any other grade of chitosan can be used here. The suspension is mixed well by stirring at 100 rpm for 30 min. The mixture is heated to 60 degree C. Formaldehyde 750 ml is then added over nominal 1-2 min. Phosphorus acid (H3PO3) 750 gm is dissolved in 7500 ml of water and added drop-wise to this mixture by stirring for 1 hour. The temperature is maintained between 60-65 degree C and preferably at 63 degree C for 6 hours after the addition is complete. The suspension is allowed to cool and filtered and the solid is air dried. The free flowing powder - small clumps are formed. They are broken mechanically and sieved using 100 mesh. The free flowing powder is then further dried in hot air oven at 70 degree C for 3 - 6 hours. Solubility of the free flowing particles is checked by adding 100 mg powder slowly to 100 ml distilled water (pH 6.5) over 5 min. The mixture is stirred continuously during addition and further for 10 more min. and then the solubility is

'checked visibly. The solubility is also checked at pH 8, 9, and 10 adjusted using dilute NaOH, or Tris buffer.
The product dissolved in water, but precipitated out when tPe pH was raised to 7.0. The dry powder obtained in this reaction was resuspended in 20 volumes of 90% methanol, and 2 volumes of concentrated HC1 were added. The mixture was stirred for one hour at room temperature. The powder was filtered out, washed with 20 vol 90% methanol 3 times and dried as described above. The resultant product was soluble from pH 3 to 12.
The products of all other examples - particularly example 10, 12 when treated with HC1 as described in this example did not alter the maximum pH of splubility.
EXAMPLE 19: USE OF WATER SOLUBLE CHITOSAN AS CARRIER.
1 gm of water soluble chitosan as derived by example 10, was dissolved in 100 ml water. To 9 ml of this solution 1 ml of copper sulfate solution (1%) was added. This was hand mixed for 2 min. Then 1 ml liquid ammonia was added. This resisted in precipitation of chitosan. Along with chitosan the bound copper sulfate is also precipitated. Copper sulfate is freely soluble in liquid ammonia and the resulting solution is deep blue in color. Estimation of copper is done by colorimetry, and using a standard graph 0.02 - 0.1%. From this the chitosan bound copper was estimated to be 90%.