DESC:Field of Invention:
The current invention is in field of, odour management formulations which maintains hygiene and ambience. More particularly, the current invention relates to a bio-based ¬ odour management formulation containing natural components that are biodegradable, eco-friendly, safe to humans, animals and environment.

Background of the Invention:
Odour is one of the most common causes of disruption of a good ambience in indoor spaces. Odour emanation is very closely linked to surface hygiene and is deeply rooted in accumulation of organic residue (human and food waste) and result in microbial growth. A common issue with managing these problems is they are masked rather than treated at the root. Most odour management solutions only contain heavy perfumes that temporarily mask the odour. Heavy usage of these products causes indoor pollution and a high incidence of respiratory Illnesses.

Due to rising awareness about these issues, these products are now being shunned by health conscious and eco-sensitised customers. As a result of these, there has been a renewed interest in replacing these chemicals, that have temporary benefits and long term ill effects, with potent natural solutions that can find the root source of the problem and have long term benefits of usage.

Currently, a plethora of products have been devised based on natural products. However, the emphasis of these developments has been in cosmetic field. The field of eco-friendly and natural odour management solutions for maintaining hygiene and ambience has largely remained untouched.

Nonetheless, there have been few ventures in this space, for instance US9451763, assigned to Laboratoire M2, discloses an aqueous disinfectant formulation containing phenolic compounds derived from natural oils and surfactants and sequestering agents. However, the product of US ‘763 does not appear to be purely natural product. The disinfectant solution invariably uses various surfactants, solvents and chelating or sequestering agents. Further, the main active of the disinfectant is a phenol extracted from natural oil, instead of oil, as such. Thus, the product as covered in US ‘763 cannot be considered as purely natural product and, since, the product uses extracts of essential oils, the process involved is costly. Furthermore, the aqueous disinfectant solution of US ‘763 cannot be considered as eco-friendly product, since it is not completely biological derived product. The formulation also does not contain enzymes and biopolymers as in the present invention, and thereby not designed with the aim of odour prevention, biofilm prevention and reduced water usage,

Another relevant disclosure could be found in US7763575, assigned to Cleanwell LLC, covers an essential oil-based cleaning and disinfecting composition containing thyme oil and a transition metal salt. Further, the formulation of US ‘575 uses a transition metal salt as an active ingredient which cannot be considered as a natural product, similar to US ‘763.

One more instance of use of essential oil as antimicrobial agents can be found in US6346281. However, the formulation as covered in US ‘281 only contains essential oil as active, which may not be effective in all the situations. It is a known fact that the efficacy of essential oils against odour and odour causing microbes is not the same across all the types of essential oils available. Sole application of essential oils is expensive, impractical and not always effective.

In view of above, it appears that this area has not been sufficiently explored.

One more consideration would be that, the currently available formulations do not consider the possible detrimental effect on the skin of the person handling these solutions. Considering that floor cleaning and other cleanings are carried out by menial labourers. These labourers use these cleaning liquids by their bare hands, so a consideration on protecting skin of handler is also a valid consideration.

Therefore, the current inventors propose a novel, eco-friendly odour prevention and hygiene maintenance formulation containing essential oil together with other biodegradable ingredients.

Summary of the Invention:
In main aspect, the invention provides an a bio-based, eco-friendly odour management formulation containing an essential oil, a bio-surfactant an emollient and a film forming agent and optionally an enzyme of biological origin.

In accordance with above aspect, all the components of the odour prevention formulation are biodegradable and natural.

The odour prevention formulation in accordance with the current invention is skin-friendly. The odour prevention formulation in accordance with the current invention does not produce fumes, is nontoxic and non-corrosive to all surfaces. It does not have any environmental impact due to its rapid biodegradability and does not leave any residue. The formulation has been created to ensure no additional water usage in some application scenarios and negligible water usage in other scenarios.

The odour management formulation of the current invention disrupts biofilm formed on the surfaces and prevents reformation of biofilm by forming a film over the surface.

In another aspect, the odour management formulation of the current invention kills malodour by neutralising odour forming molecules and prevents malodour formation by deactivating odour-causing microbes and enzymes that microbes produce which contribute to odour formation.

In yet another aspect the current invention provides a process for manufacturing the odour management formulation of the invention.

Detailed Description of Invention:
In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood by the person of ordinary skill in the art to which this invention pertains.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may not only mean “one”, but also encompasses the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

In an embodiment, the current invention describes an eco-friendly and user-friendly odour preventing formulation comprises an essential oil, a bio-surfactants, an emollient and a film forming agent and optionally a suitable enzyme of biological origin.

In an embodiment, odour preventing formulation comprises a blend of essential oils in an amount of 0.01% to 5%, a blend of bio-surfactant(s) in an amount of 0.01 to 10%, an emollient in an amount of 0.01 to 1%; and a film forming agent in sufficient quantity of biological origin.

The odour management formulation, in accordance with above embodiment, demonstrates an excellent bacteriostatic activity against most of the known pathogens, thereby preventing their colonisation and consequently preventing biofilm formation due to sustained microbial presence on surfaces.

Further, the odour management formulation of the invention inactivates other extraneous enzymes which may contribute to odour, thereby preventing odour formation.

In accordance with above embodiment, said essential oil can be one or more selected form pine oil, eucalyptus oil, citronella oil, lemongrass oil, lime oil, lemon oil, orange peel oil, rosemary oil, clove bud oil, tea tree oil, peppermint oil, cinnamon oil, thyme oil, oils of anise, oregano oil, wintergreen oil, lavender oil, hops, wheat oil, barley oil, cedar leaf oil, cedar wood oil, fleagrass oil, geranium oil, sandalwood oil, violet oil, cranberry oil, vervain oil, gum benzoin, basil oil, fennel oil, fir oil, balsam oil, menthol, Ocmea origanum oil, Hydastis carradensis oil, Berberidaceae daceae oil, Ratanhiae and Curcuma longa oil, sesame oil, macadamia nut oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil, pimento berries oil, rose oil, bergamot oil, rosewood oil, sage oil, clary sage oil, cypress oil, sea fennel oil, frankincense oil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lime oil, mandarin oil, marjoram oil, myrrh oil, neroli oil, patchouli oil, pepper oil, black pepper oil, petitgrain oil, rose otto oil, spearmint oil, spikenard oil or vetiver oil and the like.

The bio-surfactant in accordance with the above embodiment is a natural bio-surfactant derived from plants or microbes. Said bio-surfactant could be anyone or more selected from cocamidopropylamine oxide, alkyl glucoside, sodium laurel sulphate, polysorbate-type non-ionic surfactant, sophorolipids, rhamnolipids or surfactin and the like. The bio-surfactant exhibits anti-microbial activity as well as facilitate emulsification of the essential oils present in the formulation. Further, the essential oils in combination with said bio-surfactants exhibit heightened and prolonged antimicrobial activity and odour prevention activity.

The formulation, as per above embodiment, contains the emollient which acts as skin-protecting agent. Said emollient can be any one or more selected from extra virgin coconut oil, plant-based glycerol and its derivatives and the like.

The film forming agent is a natural polymer which forms a protective film over the surface. Hence, the film forming agent provides a sustained protection till the film breaks due to regular usage. However, the film formation substantially reduces the frequency of cleaning, thereby reducing the cost to the final user. Said film forming agent could be selected from chitosan, alginate or gums and the like.

The composition according to this embodiment may optionally comprise enzymes of biological origin. Said enzymes of biological origin may be selected from the group consisting of protease, amylase, pectate lyase, mannanase, lipase, cellulase and the like.

In subsequent embodiment, the odour management formulation further contains at least one scouring agent. Said scouring agent is preferably a natural organic acid which may be selected from citric acid, oxalic acid and the like.

In accordance with above embodiment, the odour management formulation of the invention is free of any sequestering agent and/or a preservative. The odour management formulation of the invention does not require any sequestering agent for maintaining optimum cleaning efficacy. Further, the absence of the sequestering agent makes the odour management formulation of the invention truly biodegradable. Furthermore, the odour management formulation of the invention is inherently self-preserving; therefore, there is no need to add any preservatives.

In an optional embodiment, the odour management formulation of the invention may further contain encapsulated enzymes and thickening agents.

In an embodiment, the present invention provides a process for manufacturing the odour management formulation of the invention.

In an advantageous embodiment, the odour management formulation of the invention demonstrates a heightened activity, in terms of odour masking as well as germ colonisation prevention, in vitro as compared to the individual components of the formulation.

The odour management formulation of the invention is intended for use as surface cleaning and sanitising agent. The odour management formulation of the invention can be used on various surfaces like floor (tiles, granite, marble, ceramic etc.), commode or urinals, (ceramic, porcelain etc.), kitchen, bathroom, utensils (metals, alloy, glass, porcelain, bone china etc.), other metal, plastic, glass and alloy surfaces.

EXAMPLES
The present invention is demonstrated by way of following examples. It is to be understood, for a person skilled in the art, they are not to be construed as limiting the scope of the invention in any way.

Example 1: Selection of Ingredients:

1. Essential oils:
Various essential oils, selected from pine, eucalyptus, citronella, lemongrass, teatree, rosemary, peppermint, clovebud, orangepeel, lime, lemon, spearmint, eucalyptus lemon, patchouli, neroulio, rosewood, camphor, kaffir lime leaf, wintergreen, melisa lemon balm, rosalina, mentha citrata against Staphylococcus aureus ATCC 6538, Proteus vulgaris ATCC 13315, Enterobacter aerogenes ATCC 13048, Escherichia coli ATCC 1.536, Klebsiella pneumoniae ATCC 4532, Salmonella enterica ATCC 10749, Pseudomonas aeruginosa ATCC 15442 and Candida albicans ATCC 10231 and 4 essential oils were selected based on broad spectrum activity and lower MIC values.
Essential oils MIC value(against most pathogens)
Lemongrass 0.50%
Teatree 0.25%
Spearmint 0.25%
Wintergreen 0.10%

2. Bio-surfactants

bio-surfactants selected from polysorbate 20, polysorbate 80, alkyl glucoside, lauryl aminoxide, cocamidopropylamine oxide, sodium laurel sulphate, , polyethylene glycol, cocoamidopropyl betaine, glucamide, Sodium cocyl glycinate against Staphylococcus aureus ATCC 6538, Proteus vulgaris ATCC 13315, Enterobacter aerogenes ATCC 13048, Escherichia coli ATCC 1.536, Klebsiella pneumoniae ATCC 4532, Salmonella enterica ATCC 10749, Pseudomonas aeruginosa ATCC 15442 and Candida albicans ATCC 10231 were tested for their minimum inhibitory concentration and 4 bio-surfactants were selected based on broad spectrum activity and lower MIC values.

Bio-surfactants MIC value (against most pathogens)
lauryl aminoxide 0.10%
sodium laurel sulphate 0.10%
cocamidopropylamine oxide 0.10%
Sodium cocyl glycinate 0.50%

3. Organic acids
various organic acids were tested for Minimum inhibitory concentration. Said organic acids were selected from citric acid, oxalic acid and acetic acid against Staphylococcus aureus ATCC 6538, Proteus vulgaris ATCC 13315, Enterobacter aerogenes ATCC 13048, Escherichia coli ATCC 1.536, Klebsiella pneumoniae ATCC 4532, Salmonella enterica ATCC 10749, Pseudomonas aeruginosa ATCC 15442 and Candida albicans ATCC 10231 and 2 organic acids were selected based on broad spectrum activity and lower MIC values.

Organic acids MIC value(against most pathogens)
Oxalic acid 0.10%
Acetic acid 0.10%

4. Bio polymers
Various biopolymers were tested for Minimum inhibitory concentration, selected from chitosan, carboxy methyl cellulose, alginate and gorghum against Staphylococcus aureus ATCC 6538, Proteus vulgaris ATCC 13315, Enterobacter aerogenes ATCC 13048, Escherichia coli ATCC 1.536, Klebsiella pneumoniae ATCC 4532, Salmonella enterica ATCC 10749, Pseudomonas aeruginosa ATCC 15442 and Candida albicans ATCC 10231 and selected chitosan based on broad spectrum activity and lower MIC value.

Biopolymer MIC value(against most pathogens)
Chitosan 0.05%

Example 2A: Preparation of Odor management formulation
The ingredients were selected based on the broad spectrum activities as mentioned in example 1 and different attributes to be imparted in final formulation like binding agent, emollient and enzymes.

Formulation A1: All ingredients
Formulation A2: Without essential oils
Formulation A3: Without surfactants
Formulation A4: Without organic acids
Formulation A5: Without chitosan

pH of the formulations were maintained at 4 in order to maintain the stability of the polymer.

The above formulations so prepared were evaluated for Inhibition of odour causing micro-organisms, biofilm forming micro-organisms, inactivation of odour causing enzymes, germ removal as mentioned in subsequent examples.

Example 2B:
The process for manufacturing the odour management formulation of Example 2A comprises of the following steps:

Step 1: Mixing, vigorously, all essential oils, glycerol with tween 20 and other surfactants;

Step 2: adding oxalic acid and acetic acid to the mixture in step 1;
Step 3: dissolving chitosan to the solution of step 2;
Step 4: making the volume to 100% using water, and

Step 5: adjusting the pH to 4 using 1N NaOH solution.

Example 3: Evaluation of stability of ingredients in the formulations
Formulation A1 Formulation A2 Formulation A3 Formulation A4 Formulation A5
Clear Hazy Clear Clear Clear
No precipitation Precipitation observed No precipitation No precipitation No precipitation

Formulation A2 was excluded as the formulation exhibited precipitation and haziness.

Example 4: Evaluation of different formulations
4.a Inhibition of odour causing (H2S producing) micro-organisms.
Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (SIM media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.
Results:

Inhibition of odour causing (H2S producing) micro-organisms Test culture used Formulation A1 Formulation A3 Formulation A4 Formulation A5
Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation No inhibition observed Complete inhibition at 1:1 dilution of the formulation

4.b Inhibition of odour causing (ammonia producing) micro-organisms.
Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Christensen urea media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of odour causing (ammonia producing) micro-organisms Test culture used Formulation A1 Formulation A3 Formulation A4 Formulation A5
Proteus vulgaris ATCC 13315 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation

4.c Inhibition of biofilm producing micro-organisms.
Formulations were tested against Enterobacter cloacae ATCC 11439 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Congo red broth media). Concentration of 1x10^6 cfu/ml of 18 hours old Enterobacter cloacae was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of biofilm producing micro-organisms Test culture used Formulation A1 Formulation A3 Formulation A4 Formulation A5
Enterobacter cloacae ATCC 11439 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation

4.d Inactivation of odour causing enzyme.
Formulations were tested against Urease enzyme extracted from Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Christensen urea media). 0.1ml of urease containing broth extracted from Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inactivation of odour causing enzymes Test enzyme used Formulation A1 Formulation A3 Formulation A4 Formulation A5
Urease from Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation

4.e Time based germ removal.
Formulations were tested against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442.

Formulations were inoculated with a concentration of 1x10^7 cfu/ml of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 in 1:2 ratio with contact time of 15 minutes. Reduction in cfu/ml was done by spread plate technique (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count). Respective controls, positive control (only culture suspension) and negative control (1:2 diluted using sterils water) were kept.

Results:
Time based germ removal Test culture used Formulation A1 Formulation A3 Formulation A4 Formulation A5
Staphylococcus aureus ATCC 6538 99% reduction 87% reduction 99% reduction 99% reduction
Pseudomonas aeruginosa ATCC 15442. 99% reduction 88% reduction 99% reduction 99% reduction

4.f Formation of protective film on the surface:
Formulations were examined for the formation of a protective film on the surface of glass. Slides were sprayed using formulations and allowed to dry. The film forming capacity was evaluated on basis of stain retained on the slide after washing. Staining was done using crystal violet stain.
Film formation Control Formulation A1 Formulation A3 Formulation A4 Formulation A5
+ +++ +++ +++ +

+ indicates intensity of the stain retained after wash.

Example 5: Preparation of formulations with varied concentrations of ingredients used
5.1 Decreased concentrations of ingredients by 25%

5.1 Increased concentrations of ingredients by 25%
Example 6: Evaluation of formulation with decreased concentrations of ingredients.

6.a Inhibition of odour causing (H2S producing) micro-organisms.
Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (SIM media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of odour causing (H2S producing) micro-organisms Test culture used Formulation A1 Formulation A1.0 Formulation A1.1 Formulation A1.2 Formulation A1.3
Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation

6.b Inhibition of odour causing (ammonia producing) micro-organisms.
Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Christensen urea media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of odour causing (ammonia producing) micro-organisms Test culture used Formulation A1 Formulation A1.0 Formulation A1.1 Formulation A1.2 Formulation A1.3
Proteus vulgaris ATCC 13315 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:3 dilution of the formulation Complete inhibition at 1:3 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation

6.c Inhibition of biofilm producing micro-organisms.
Formulations were tested against Enterobacter cloacae ATCC 11439 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Congo red broth media). Concentration of 1x10^6 cfu/ml of 18 hours old Enterobacter cloacae was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of biofilm producing micro-organisms Test culture used Formulation A1 Formulation A1.0 Formulation A1.1 Formulation A1.2 Formulation A1.3
Enterobacter cloacae ATCC 11439 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:1 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation
6.d Inactivation of odour causing enzyme.
Formulations were tested against Urease enzyme extracted from Proteus vulgaris ATCC 13315 at different dilutions.

Different dilutions of the formulations were made in sterile culture media (Christensen urea media). 0.1ml of urease containing broth extracted from Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inactivation of odour causing enzymes Test culture used Formulation A1 Formulation A1.0 Formulation A1.1 Formulation A1.2 Formulation A1.3
Urease from Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:6 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation

6.e Time based germ removal.

Formulations were tested against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442.

Formulations were inoculated with a concentration of 1x10^7 cfu/ml of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 in 1:2 ratio with contact time of 15 minutes. Reduction in cfu/ml was done by spread plate technique (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count). Respective controls, positive control (only culture suspension) and negative control (1:2 diluted using sterils water) were kept.
Results:
Time based germ removal Test culture used Formulation A1 Formulation A1.0 Formulation A1.1 Formulation A1.2 Formulation A1.3
Staphylococcus aureus ATCC 6538 99% reduction 94% reduction 92% reduction 95% reduction 99% reduction
Pseudomonas aeruginosa ATCC 15442. 99% reduction 86% reduction 94% reduction 93% reduction 98% reduction

Example 7: Evaluation of formulation with increased concentrations of ingredients.

7.a Inhibition of odour causing (H2S producing) micro-organisms.

Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.
Different dilutions of the formulations were made in sterile culture media (SIM media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.
Results:
Inhibition of odour causing (H2S producing) micro-organisms Test culture used Formulation A1 Formulation A1.4 Formulation A1.5 Formulation A1.6 Formulation A1.7
Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:12 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:12 dilution of the formulation
7.b Inhibition of odour causing (ammonia producing) micro-organisms.
Formulations were tested against Proteus vulgaris ATCC 13315 at different dilutions.
Different dilutions of the formulations were made in sterile culture media (Christensen urea media). Concentration of 1x10^6 cfu/ml of 18 hours old Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of odour causing (ammonia producing) micro-organisms Test culture used Formulation A1 Formulation A1.4 Formulation A1.5 Formulation A1.6 Formulation A1.7
Proteus vulgaris ATCC 13315 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation

7.c Inhibition of biofilm producing micro-organisms.

Formulations were tested against Enterobacter cloacae ATCC 11439 at different dilutions.
Different dilutions of the formulations were made in sterile culture media (Congo red broth media). Concentration of 1x10^6 cfu/ml of 18 hours old Enterobacter cloacae was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inhibition of biofilm producing micro-organisms Test culture used Formulation A1 Formulation A1.4 Formulation A1.5 Formulation A1.6 Formulation A1.7
Enterobacter cloacae ATCC 11439 Complete inhibition at 1:5 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:8 dilution of the formulation Complete inhibition at 1:5 dilution of the formulation

7.d Inactivation of odour causing enzyme.

Formulations were tested against Urease enzyme extracted from Proteus vulgaris ATCC 13315 at different dilutions.
Different dilutions of the formulations were made in sterile culture media (Christensen urea media). 0.1ml of urease containing broth extracted from Proteus vulgaris was inoculated in respective dilutions. Respective positive control (only culture media) and negative control (dilutions using sterile water) were kept. All samples were vortexed and incubated overnight at 37°C.

Results:
Inactivation of odour causing enzymes Test culture used Formulation A1 Formulation A1.4 Formulation A1.5 Formulation A1.6 Formulation A1.7
Urease from Proteus vulgaris ATCC 13315 Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation Complete inhibition at 1:10 dilution of the formulation
7.e Time based germ removal.
Formulations were tested against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442.
Formulations were inoculated with a concentration of 1x10^7 cfu/ml of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 in 1:2 ratio with contact time of 15 minutes. Reduction in cfu/ml was done by spread plate technique (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count).. Respective controls, positive control (only culture suspension) and negative control (1:2 diluted using sterils water) were kept.

Results:

Time based germ removal Test culture used Formulation A1 Formulation A1.4 Formulation A1.5 Formulation A1.6 Formulation A1.7
Staphylococcus aureus ATCC 6538 99% reduction 99% reduction 99% reduction 99% reduction 99% reduction
Pseudomonas aeruginosa ATCC 15442. 99% reduction 99% reduction 99% reduction 99% reduction 99% reduction

Example 8: Evaluation of selected formulation (A1)

8.a Evaluation of formulation for disruption of existing biofilm and prevention of biofilm formation

Formulation was tested against pathogenic biofilm forming culture Enterobacter cloacae ATCC 11439.
An invitro biofilm of Enterobacter cloacae ATCC 11439 was produced on glass slide. Biofilm produced was confirmed by Crystal violet staining technique and count of the same was also noted.
For disruption of the existing biofilm, formulation was sprayed over the surface of biofilm, with water sprayed as control and allowed to dry for 15minutes and plating of both the samples was done to check for reduction in count (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count).
Results:

Disruption of existing biofilm present Samples Test culture used Initial count
(cfu/ml) Count after treatment (cfu/ml) % reduction in count
Control Enterobacter cloacae ATCC 11439 1x107 5x106 50.00%
Test 1x107 4x103 99.90%

For prevention of biofilm formation, glass slide was exposed to Enterobacter cloacae ATCC 11439 (germ) at concentration of 1x10^6 cfu/ml followed by treatment with formulation and water for test and control respectively. The same step was repeated 3 times. Prevention of biofilm formation was evaluated on basis of plate count (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count) before and after germ exposure.
Results:
Samples Test culture used Initial count
I germ exposure (cfu/ml) Count after I treatment
(cfu/ml) Count after II germ exposure (cfu/ml) Count after II treatment (cfu/ml) Count after III germ exposure (cfu/ml) Count after III treatment (cfu/ml)
Control Enterobacter cloacae ATCC 11439 1x105 5x104 2x106 6x105 1x106 5x105
Test 4x104 4x103 1x103 1x102 1x102 1x102
8.b Evaluation of formulation for protective film formation:
For film formation, glass slide was sprayed with the formulation (test) and water(control) and dried followed by exposure of Enterobacter cloacae ATCC 11439 (germ) at concentration of 1x10^6 cfu/ml. The slide was exposed to germ repeatedly 3 times. Protective film formation was evaluated on basis of plate count (Bacteriological Analytical Manual, Chapter 3; Aerobic Plate Count) after every germ exposure.
Results:
Samples Test culture used Initial count
No germ Count after I germ exposure (cfu/ml) Count after II germ exposure (cfu/ml) Count after III germ exposure (cfu/ml)
Control Enterobacter cloacae ATCC 11439 2x101 5x105 2x106 6x106
Test NIL 1x101 2x101 1x101

Example 9:
9.a Evaluation of formulation for neutralization and masking of odour

Formulation was tested for masking of odour against bad odour present in urinals.
Results were evaluated on basis of hedonic perception of all individuals.

Total number of persons = 10
Average Rating for dislike of odour = -1.7 (this falls in category of DISLIKE VERY MUCH)
Average intensity of bad odour = 3.5 (Clearly perceivable to strong)
Average rating of liking of odour = +0.9 (Falls in category of like slightly/ pleasing odour)
Average intensity of pleasant smell = 3.6 (clearly perceivable to strong)
There is a shift from bad odour to pleasing odour / the bad odour is masked using the formulation

9.b Evaluation of formulation for neutralization and masking of adour
Ratings for hedonic tone scale before application of the formulation:
Number of persons Hedonic tone Value for tone Intensity Value for intensity
1 Extremely dislike -3 Very strong 5
2 Extremely dislike -3 Very strong 5
3 Dislike very much -2 Clearly perceivable 3
4 Extremely dislike -3 Very strong 5
5 Dislike moderately -1 Strong 4
6 Dislike moderately -1 Clearly perceivable 3
7 Dislike moderately -1 Faintly perceivable 2
8 Dislike moderately -1 Clearly perceivable 3
9 Dislike moderately -1 Faintly perceivable 2
10 Dislike moderately -1 Clearly perceivable 3
Average rating Falls between dislike moderately to dislike very much category -1.7 Falls between clearly perceivable to strong category 3.5

Ratings on hedonic tone scale after application of the formulation:
Number of persons Hedonic tone Value for tone Intensity Value for intensity
1 Neither like nor dislike 0 Faintly perceivable 2
2 Like slightly/Pleasant 1 Clearly perceivable 3
3 Like slightly/Pleasant 1 Clearly perceivable 3
4 Like slightly/Pleasant 1 Strong 4
5 Like slightly/Pleasant 1 Strong 4
6 Like slightly/Pleasant 1 Strong 4
7 Like slightly/Pleasant 1 Strong 4
8 Like slightly/Pleasant 1 Strong 4
9 Like slightly/Pleasant 1 Strong 4
10 Like slightly/Pleasant 1 Strong 4
Average rating Falls towards the category of pleasant and like slightly 0.9 Falls between clearly perceivable to strong category 3.6
Reference: Lim J, Hedonic scaling: A review of methods and theory, Food Quality and Preference 22 (2011) 733–747

9.c Evaluation of formulation for biodegradability test
The biodegradability test of the formulation was carried out under aerobic conditions. Biodegradability was measured on basis of reduction in Chemical Oxygen Demand. Formulation was subjected to treatment with aerobic microbial consortia and the oxygen demand was evaluated every 7 days.
Results:
Biodegradability COD readings (ppm)
Day 1 Day 8 Day 15 Day 21 Day 28
707 450 304 124 39

COD was reduced by 94.4% at the 28th day of the test.
Example 10: Preparation of Odour management formulation containing enzymes

Enzyme blend is a commercial blend of liquid enzymes containing multiple hydrolytic enzymes.
,CLAIMS:
1. A bio-based, eco-friendly odour management formulation comprising;
a. an essential oil;
b. a bio-surfactant;
c. an emollient; and
d. a film forming agent.

2. The bio-based odour management formulation as claimed in claim 1, wherein the odour formulations are biodegradable and natural.

3. The bio-based odour management formulation as claimed in claim 1, wherein the essential oil is selected from, but not limited to, pine oil, eucalyptus oil, citronella oil, lemongrass oil, lime oil, lemon oil, orange peel oil, rosemary oil, clove bud oil, tea tree oil, peppermint oil, cinnamon oil, thyme oil, oils of anise, oregano oil, wintergreen oil, lavender oil, hops, wheat oil, barley oil, cedar leaf oil, cedar wood oil, fleagrass oil, geranium oil, sandalwood oil, violet oil, cranberry oil, vervain oil, gum benzoin, basil oil, fennel oil, fir oil, balsam oil, menthol, Ocmea origanum oil, Hydastis carradensis oil, Berberidaceae daceae oil, Ratanhiae and Curcuma longa oil, sesame oil, macadamia nut oil, evening primrose oil, Spanish sage oil, Spanish rosemary oil, coriander oil, pimento berries oil, rose oil, bergamot oil, rosewood oil, sage oil, clary sage oil, cypress oil, sea fennel oil, frankincense oil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lime oil, mandarin oil, marjoram oil, myrrh oil, neroli oil, patchouli oil, pepper oil, black pepper oil, petitgrain oil, rose otto oil, spearmint oil, spikenard oil or vetiver oil and the like.

4. The bio-based odour management formulation as claimed in claim 3, wherein the essential oil is in an amount of 0.01% to 5%.

5. The bio-based odour management formulation as claimed in claim 1, wherein the bio-surfactant is selected from, but not limited to, cocamidopropylamine oxide, alkyl glucoside, sodium laurel, sulphate, polysorbate-type non-ionic surfactant, sophorolipids, rhamnolipids or surfactin and the like.

6. The bio-based odour management formulation as claimed in claim 5, wherein the bio-surfactant is in an amount of 0.01 to 10%.

7. The bio-based odour management formulation as claimed in claim 1, wherein the emollient is selected from, but not limited to, virgin coconut oil, plant-based glycerol and its derivatives and the like.

8. The bio-based odour management formulation as claimed in claim 7, wherein the emollient is in an amount of 0.01 to 1%.

9. The bio-based odour management formulation as claimed in claim 1, wherein the film forming agent is selected from, but not limited to, chitosan, alginate or gums and the like.
10. The bio-based odour management formulation as claimed in claim 1, further contains a scouring agent.

11. The bio-based odour management formulation as claimed in claim 10, wherein the scouring agent, is a natural organic acid.

12. The bio-based odour management formulation as claimed in claim 11, wherein the natural organic acid is selected from citric acid, oxalic acid and the like.

13. The bio-based odour management formulation as claimed in claim 1, wherein the formulation optionally further contains an enzyme.

14. The bio-based odour management formulation as claimed in claim 13, wherein the enzyme is of biological origin.

15. The bio-based odour management formulation as claimed in claim 14, wherein the enzymes is selected from the group consisting of protease, amylase, pectate lyase, mannanase, lipase, cellulase and the like.

16. A process for manufacturing the odour management formulation as claimed in claim 1, wherein the process comprises the following steps;
Step 1: Mixing, vigorously, all essential oils, glycerol with tween 20 and other surfactants;
Step 2: adding oxalic acid and acetic acid to the mixture in step 1;
Step 3: dissolving chitosan to the solution of step 2;
Step 4: making the volume to 100% using water, and
Step 5: adjusting the pH to 4 using 1N NaOH solution.