Description:
Field of Invention:
The present invention relates to preservative system for personal care compositions. More particularly, the present invention relates to preservative system comprising N-acyl L-proline and N-acyl L-glutamine that are prebiotic towards skin microbiota and the preservative system is cold-processable. Further the invention also discloses a method of preventing microbial contamination of personal care compositions.

Background and prior art of the invention:
Preservation of personal care products continues to be a challenge because the antimicrobials of yesteryears have been found to be dangerously toxic to both humans and ecology. Toxic effects of these diverse antimicrobials (for ex. halogenated molecules, isothiazolinones, parabens, formaldehyde releasers etc.) range from allergic reactions to neurotoxicity and carcinogenicity. In view of this serious toxicity associated with most of ‘effective’ antimicrobials of yesteryears, the personal care industry is trying to replace the toxic ones with the gentler antimicrobials (phenoxy ethanol, benzoic acid, sorbic acid, dehydroacetic acid, benzyl alcohol etc.; Cosmetics Directive Annex VI, https://echa.europa.eu/cosmetics-preservatives). However, the biggest concern about these gentle antimicrobials is that they all are based on fossil fuel and bio-based carbon content varies from 0 % to at best 60 %. This low % of bio-based carbon (and high content of fossil fuel-based carbon) is totally undesirable from the perspective of the ‘sustainability’ of planet. A recent patent application (Indian patent application, IN 202421009600, published on 29th March 2024) covers the detailed account of the serious limitations of these antimicrobials and reports an alternative to fossil fuel-based antimicrobials. It proposes a combination of gentle antimicrobials that are bio-renewable/bio-resourced, namely, N-capryloyl L-glutamine and glyceryl mono undecylenate.
This combination of N-capryloyl L-glutamine with glyceryl mono undecylenate of IN 202421009600 is ‘biodegradable’ and is totally based on bio-renewable feed stock, however, there are a few serious drawbacks associated with it.
The first major drawback is the in-solubility of glyceryl mono undecylenate in water. This creates serious limitations in creating transparent personal care compositions such as face wash or body wash which are generally perceived by consumers as pure due to transparency. At small dosage level, it does get solubilized in surfactant systems of a typical body wash or a shampoo but higher dosage level it imparts aesthetically unpleasant hazy appearance. Sometimes it shows up in transparent formulations after a few days of standing at room temperature. These limitations are due to water-insolubility of this fatty acid ester which is typically a mixture of mono (~ 50 %), di and triglycerides of undecylenic acid.
The second drawback of the above-mentioned invention is that the two antimicrobials need to be added separately. It should be noted that N-acyl L-glutamines are solids with melting points above 100 ?. These cannot be combined together to solubilize solid N-acyl L-glutamines in liquid glyceryl undecylenate since highly acidic carboxyl group of N-acyl L-glutamine reacts with the hydroxyl groups of glyceryl mono undecylenate in acid catalyzed esterification and generates corresponding impurities. Also, the blend of N-acyl glutamine and glyceryl mono undecylenate shows immediate discoloration at room temperature. Glyceryl mono undecylenate’s liquid nature can’t be exploited to disperse N-acyl L-glutamine since both are incompatible with each other and deteriorate in color very rapidly. The color deterioration is due to the unsaturation of undecylenoyl chain. Hence, the two ingredients, namely, N-acyl L-glutamines and glyceryl undecylenate, need to be added separately and for the dissolutions of high melting N-acyl L-glutamines, whole personal care compositions need to be heated up. This lack of processing facility at room temperature for personal care formulations that are manufactured at a very a large scale (in tens of metric tonne scale) results in spending a lot of energy in heating the whole mass of the formulation. The highly polar and reactive nature of N-acyl L-glutamines (reactive carboxyl group) makes it difficult to dissolve them in any other liquid personal care ingredient of small volume, that can be later added to be main bulk of a personal care formulation. N-Acyl L-glutamine can’t be dissolved in other liquid personal care ingredients like humectant glycols or emollients etc. however, during this operation the highly reactive carboxylic group of acyl glutamine reacts with hydroxyl or amino or ester group of these ingredients to form corresponding amides, esters or salts (with tertiary amines) or other trans-esterified products.
N-Capryloyl L-glutamine of the patent application IN 202421009600 is a solid hydrophobic antimicrobial with a high melting point. Incorporating it into personal care compositions at room temperature (cold temperature processibility) is a challenge.
Hence, it is obvious that the ‘green’ antimicrobial combination of IN 202421009600, is still falling short of fulfilling the expectations of an ‘ideal’ preservation system that can be green, efficacious and cold processable and with additional personal care benefits to the user.
Therefore, there remains a challenge to provide a cold processable, broad spectrum preservation system that is completely based on bio renewable raw materials, biodegradable and at the same time it’s suitable for both transparent and opaque personal care compositions.
There also remains a need for the preservative system that are chemically stable and does not discolor the formulation during the shelf life and should be compatible with other ingredients.
Yet further the preservative system shall meet all the four requirements of an ‘ideal’ preservative listed below:
1) efficacious broad-spectrum antimicrobial (active against bacteria, yeast and mold) 2) biodegradable without harming the ecology, 3) manufactured by processes that meets all twelve principles of ‘Green’ chemistry and engineering (Anastas and Warner, Green Chemistry: Theory and Practice New York, Oxford University Press, 1998) and 4) totally based on ‘bio-renewable’ starting materials for long term sustainability.

Objectives of the invention
i. It is an objective of the present invention to create cold-processable, biodegradable preservation system based on bio-renewable feed stocks for personal care compositions.
ii. Another objective of the present invention is to create a preservative system for personal care compositions that would be based on antimicrobials which are prebiotic towards the human skin microbiota.
iii. Another objective of the present invention is to create a broad-spectrum antimicrobial preservative system for personal care compositions that are effective at pH range of 3.0 to 6.0.
iv. Another objective of the present invention is to create an organoleptically and aesthetically acceptable preservative system for personal care compositions that would be compatible with commonly used personal care ingredients.
v. Yet another objective of the present invention is to create a preservation system for personal care compositions avoiding use of petrochemicals, hazardous chemicals and hazardous processing conditions at any stage of synthesis or processing.
vi. Further objective of the present invention is to create an antimicrobial preservative system for personal care compositions that would avoid antimicrobial molecules with any functional group with a potential to be cytotoxic like phenolic moiety, halogens (chlorine, bromine, iodine), ‘difficult-to-biodegrade’ heterocycles and formaldehyde releasing functionalities etc.

Summary of the Invention:
In an aspect, the present invention relates to a cold-processable, bio-renewable, biodegradable and prebiotic preservative system for personal care compositions, comprising:
i. N-acyl L-proline of Formula I wherein R1CO is selected from fatty acyl of C8 to C18;
ii. N-acyl L-glutamine of Formula II wherein R2CO is selected from capryloyl or undecylenoyl;

Formula I

Formula II

wherein the molar ratio of N-acyl L-proline of Formula I, and N-acyl L-glutamine of Formula II, is from 2:1 to 1:2 and
iii. water; comprising of 20 to 30 %by weight of the total preservative system;
wherein the both N-acyl L-amino acids are manufactured in one pot sequentially by reacting fatty acid chlorides with the corresponding amino acids in the presence of base.
In another aspect, the present invention provides a personal care composition comprising:
i. N-acyl L-proline of Formula I wherein R1CO is selected from fatty acyl of C8 to C18;
ii. N-acyl L-glutamine of Formula II wherein R2CO is selected from capryloyl or undecylenoyl; and
iii. one or more personal care ingredient,
In an embodiment the personal care composition of the present invention is devoid of petrochemical based anti-microbial as preservative.
In another embodiment the personal care compositions, preserved with combinations of antimicrobials of the present invention, have pH ranging from 3.0 to 6.0.
In yet another embodiment the personal care composition is selected from shampoo, body wash, face wash, hair conditioner, hair serum, soap bar, syndet bar, cream, lotion, hygiene wash, deodorant, anti-perspirant and gel.
In another aspect the present invention provides a method of preventing microbial contamination of a personal care composition, said method comprises the addition of 0.5 to 4 % by weight, based on the total weight of the composition, of at least one compound of Formula I and at least one compound of Formula II,

Formula I

Formula II

wherein R1 of Formula I is selected from fatty acyl of C8 to C18 and R2 in Formula II is selected from capryloyl and undecylenoyl.

In an embodiment a method of preventing microbial contamination of a personal care composition wherein the personal care composition is selected from shampoo, body wash, face wash, hair conditioner, hair serum, soap bar, syndet bar for hair and skin, cream, lotion, hygiene wash, deodorant, anti-perspirant and gel.
In yet another embodiment a method of preventing microbial contamination of a personal care composition wherein the personal care composition has pH of between 3 to 6.
In yet another embodiment a method of preventing microbial contamination of personal care compositions wherein ratio of the compound of formula I and the compound of formula II is between 1:2 and 2:1.
Brief description of Figures:
Figure 1 depicts the effect on growth of S. aures and S. epidermidis in the petri plates with treatment (test) and without treatment (control) of the preservative system of present invention to demonstrate prebiotic property.
Detailed Description of the Invention:
The term “cold-processable” as used in the specification refers to ability of the preservative system of present invention to be processable at room temperature or at lower temperature. It also substantially means that the preservative system can be added to personal care composition without requirement of heat treatment.
The term “bio-renewable” as used in the specification refers to the raw materials, used in preparation of preservative system of the present invention, are derived from bio-renewable sources as per ISO 16128 guidelines.
The term biodegradable as used in the specifications refers to the ability of the preservative system of the present invention to be able to be broken down into environmentally safe components.
The term “prebiotic” as used in the specification refers to the property of the preservative system of the present invention to promote the healthy balance of microbiome of human skin by supporting the beneficial bacteria that oppose colonization of pathogenic bacteria.
As discussed in the background section, all those powerful traditional antimicrobials for preservation of personal care products are reported to be toxic to human beings and in some cases, they are reported to be toxic to other forms of life in the environment. All powerful antimicrobials of yesteryears are implicated in toxicity issues ranging from allergies to neurotoxicity to carcinogenicity. Many are reported to disrupt endocrine systems. Personal care industry is consciously phasing the toxic antimicrobial preservatives (Examples, chloromethyl isothiazolinone, Triclosan, parabens, DMDM Hydantoin, and Quaternium-15) out of products for consumers. However, alternative biodegradable efficacious preservatives, produced by ‘green’ processes and derived from bio-renewable feedstock, are still not available.
This problem of eco-friendly and efficacious antimicrobial preservation system for personal care products has been addressed to some extent by Koshti et al. (IN 202421009600) by deploying a combination of a novel ‘green’ antimicrobial, N-capryloyl L-glutamine with glyceryl mono undecylenate. The combination of the two exhibits synergy regarding antimicrobial activity and both molecules are derived from bio-renewable feedstock and are biodegradable. In combination, these two molecules do an adequate job of preservation, however, there are a few limitations while deploying the combination in all types of personal care compositions. N-capryloyl L-glutamine is not a cold- processable antimicrobial. It is not only a water-insoluble solid, but it melts 122 ?. Hence ease of incorporation of this high melting and water-insoluble product in flake form at ambient temperature is a major hurdle. Glyceryl mono undecylenate is liquid but it cannot be used to dissolve or disperse N-capryloyl L glutamine since these two molecules react with each other if mixed and leading to formation of new entities and severe brownish coloration. Water-insolubility of glyceryl mono undecylenate restricts the application of this combination for opaque personal care compositions only. Almost all products and molecules derived from undecylenic acid have problems of color deterioration during the synthesis as well as on standing. This problem is more acute with undecylenic acid derivatives that are liquid. Glyceryl mono undecylenate is colored product (color 3 to 5 on Gardner scale) generated during synthesis) and it keeps on deteriorating during storage due to both heat and light sensitivity.
In the present invention, the inventors have remedied the problems and challenges described in the background section by using a combination of bio-sourced, biodegradable and cold-processable N-acyl derivatives of natural L-amino acids to preserve both opaque and transparent compositions with a pH range of 3.0 to 6.0. The present invention provides a combination of green antimicrobial preservative system comprising of N-acyl L-proline (Formula I wherein, R1CO = C8 to C18) with N-acyl L-glutamine (Formula II, R2CO is either capryloyl or undecylenoyl).
N-Acyl L-prolines
N-Acyl L-prolines (CAS No, 58725-39-6) are known to the personal care industry. N-Acyl L-prolines of the present invention are made from fatty acids derived from bio-renewable source and L-proline. L-Proline is one of the twenty natural amino acids which is an important component of human collagen as well as part of cardiac muscles. This is biosynthesized by humans and other mammals from L-glutamic acid, another non-essential amino acid. L-Proline is especially important in the production of collagen, a structural protein which is a primary component of skin, cartilage and bone. L-Proline is taken as dietary supplement and is available over the counter.
Potassium N-acyl L-prolinates have been used as emulsifiers (US Patent 11771643) and as hair and skin conditioning agents. N-Acyl L-prolines and N-acyl L-prolinates are part of ready concoctions that are offered for multiple skin or hair benefits, for examples, SEPICALM VG WP (Sodium palmitoyl prolinate with Nymphaea alba flower extract, for anti-inflammatory, soothing and calming effect) and SEPIFEEL™ ONE is a composition for texturing the formulations (palmitoyl proline, magnesium palmitoyl glutamate and sodium palmitoyl sarcosinate).
Garnier uses SEPICALM VG WP in soap based facial cleanser containing hyaluronic acid and the whole composition is preserved with sodium benzoate and phenoxy ethanol. Another moisturizer by Garnier with SPF 15 contains sodium palmitoyl prolinate and is preserved with phenoxy ethanol. Moisturizing gel from Garnier also deploys SEPICALM VG WP and is preserved with iodopropynyl butyl carbamate (IPBC). A similar combination of SEPICALM VG WP and IPBC is also present in VICHY’s deodorant. Thus, palmitoyl prolinate (CAS No 58725-33-0) is deployed in a variety of personal care formulations that are preserved with antimicrobial preservatives like phenoxy ethanol and IPBC.
Another gentle cleanser that seems to be getting popular is sodium N-cocoyl L-prolinate which is part of the recently launched (2024) bio-organic cleanser by Dr Neidermaier Pharma. Also, a face wash with N-cocoyl L-prolinate, launched by Marks and Spencer (2024) is preserved with synthetic petrochemical based antimicrobials like phenoxy ethanol, and 2-ethyl glycerin. Deodorant spray from Coop, Italy, is based on cocoyl proline and preserved with antimicrobial sodium usnate that is derived from lichens. Use of N-cocoyl L-proline is also seen in another example of deodorant stick for females (Lavilin from Latvia) containing strong antimicrobials like capryloyl glycine, a quaternary ammonium chloride, sodium sorbate, sodium benzoate, and ethyl hexyl glycerin. Shower scrub using powdered olive seeds by Bioselect, Denmark, contains N-cocoyl L-proline and whole composition is preserved with antimicrobials like sodium dedydroacetate, sodium sorbate and sodium benzoate. Hand soap by De Labo has sodium lauryl sulphate and N-cocoyl L-proline and the whole composition is preserved with synthetic petrochemical based antimicrobials like sodium benzoate, and dehydroacetic acid. It is interesting to note that Weleda AG uses N- cocoyl L-proline in range of face care products like cleansers, spray and mousse. It can be noted that N-acyl L-proline has been used as skin benefit agent, but it had never been used as a stand-alone preservative or in a combination of an antimicrobial preservative systems in any personal care composition.
N-Acyl L-prolines of present invention are synthesized (Examples 1, 2 & 3) from corresponding fatty acyl chlorides and L-proline in the presence of base in aqueous medium. Acyl chlorides are made by reacting fatty acids with oxalyl chloride under the catalytic influence of the same N-acyl L-proline that is being synthesized. For example, lauroyl chloride needed for synthesis of N-lauroyl L-proline is made by reacting oxalyl chloride and lauric acid under the catalytic influence of N-lauroyl L-proline. For syntheses of N-acyl L-prolines, a variety of acyl chlorides are synthesized using corresponding fatty acids. The minimum inhibitory concentrations against a variety of microbes (Table 1) indicates that N-acyl L-prolines with longer acyl chain (C12 and above) seem to exhibit slightly better anti-microbial activity against Gram positive microbes, conversely, N-capryloyl L-proline (C8) seems to have better activity against Gram -ve bacteria. N-Capryloyl L-proline (Example 3) and N-lauroyl L-proline (Example 1) are made from single individual fatty acid, caprylic and lauric acid respectively whereas N-cocoyl L-proline is made from coco fatty acid which is mixture of C8 to C18) fatty acids (Example 2) with C12 to C18 fatty acids content being around 90 % of the total fatty acids. Typical distribution of coco fatty acid is C8, 4-8 %, C10, 4-10 %, C12, 58-65 %, C14, 14-24 % C16, 1-8 % C18, 0.5 -1.0 %.
N-Acyl L-prolines (R1CO = C8 to C18) of present invention are extremely active against the pathogenic Staphylococcus aureus. The sweat degrading resident Gram positive bacteria namely, Corynebacterium xerosis, Micrococcus luteus and Staphylococcus hominis of human axillary vault, are inhibited at very low concentration, making N-acyl L-prolines to be very effective as part of anti-malodor (deodorant) compositions. The N-acyl L-prolines of Table I (Example 4) are also very effective against the yeast Candida and bacterium Gardnerella vaginalis that are implicated in feminine intimate health and hygiene. Another interesting personal care application is scalp hygiene (seborrheic dermatitis/dandruff) wherein yeast Malassezia proliferates to aggravate.
The human body’s natural defense mechanism involves small proline-rich proteins (SPRRs) that are released in response to infection, stress or other inflammatory stimuli in gastrointestinal, respiratory, and urinary tracts (Hooper et al. Science, 291:881–884, 2001, Demetris et al. Journal of Hepatology 48:276–288, Hu et al. Science, 374:eabe 6723). Similarly, the bactericidal activities of small proline rich proteins (SPRRs) are also part of skin’s immune defense system and are induced in sebocytes by lipopolysaccharides (LPS) secreted by bacterial cell walls. (‘Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.’ Chenlu Zhang et al.; Elife 2022 Mar 2:11e76729. doi; 10.7554/6/eLife76729, an international collaboration amongst the institutes of the USA, Germany and China, namely, University of Texas Southwestern medical center, Shanghai Tech University, Brandenburg Medical School). Sebum secretion acts as a delivery system for these antimicrobial peptides that exhibit good activity against highly infectious Pseudomonas aeruginosa and Staphylococcus aureus. These antimicrobial small proline-rich proteins breach the cell membrane /cell wall of microbes. Similarly, the N-acyl L-prolines, the peptidic amphiphiles breach the cell membranes of microbes (Minimum inhibitory concentrations, Table 1).
Suitably the N-acyl L-proline of the present invention can be N-capryloyl L-proline, N-nonoyl L-proline, N-decanoyl L-proline, N- undecanoyl L-proline, N-lauryl L-proline, N-cocoyl L-proline, N-myristoyl L-proline, N-palmitoyl L-proline, N-palmitoleoyl L-proline, N-stearoyl L-proline, N-oleoyl L-proline, N-linoleoyl L-proline, N-linonenoyl L-proline.

N-Acyl L-glutamines
N-Acyl L-glutamines are deployed in combination with N-acyl L-prolines to form the preservative system of the present invention. N-Acyl L-glutamines (IN 202421009600) are made from fatty acids and L-Glutamine, an amino acid, which is a building block of proteins. The human body receives its L-glutamine from animal products like milk, eggs, meat as well as vegetables like kale, celery, beans, wheat and sprouts. In addition, human body does synthesize L-glutamine. It is needed for muscle building (growth and repair) and is linked to the immune system. It has beneficial effects in patients suffering with Sickle Cell disease. L-Glutamine is industrially manufactured from glucose by fermentation. In the present invention, L-Glutamine and fatty acids are bonded together via a peptide linkage in N-acyl L-glutamines (Formula II, R2CO is either capryloyl, undecylenoyl or mixture thereof).

Formula II
Fatty acids, saturated or unsaturated, ranging from R2CO = C8 to C18 are derived from bio-renewable sources. Unsaturated undecylenic acid is derived from ricinoleic. Fatty acids are converted into fatty acid chloride and reacted with L-glutamine in the presence of inorganic base in aqueous medium. Examples 5 and 6 demonstrate the synthesis and full characterization of N-capryloyl L-glutamine (Formula III) and N-undecylenoyl L-glutamine (Formula IV).

Formula III

Formula IV
N-Capryloyl L-glutamine and N-undecylenoyl L-glutamine exhibit decent antimicrobial properties against bacteria, yeast and mold (minimum inhibitory concentrations (MICs) listed in Table 2a, Example 7). It can be seen that N-acyl L-glutamines are useful personal-care benefit agent due its excellent inhibitory activity against Cutibacterium acnes (MIC, 0.2%) which is involved in exacerbation of Acne vulgaris, as well as against Malassezia yeast (MIC, 0.2%) which is involved in the proliferation of dandruff. It should also be noted that N-capryloyl L-glutamine has moderate activity against sweat degrading gram positive residents Staphylococcus hominis (MIC, 0.6 %) Micrococcus luteus (MIC 0.5 %) and Corynebacterium xerosis (MIC, 0.5%) of axillary vault region of human. This property is useful in personal care composition designed to control the generation of body odor.
Preservation efficacy of combinations of N-acyl L-proline and N-acyl L-glutamine
A method of preventing microbial contamination of a personal care composition is disclosed herewith. The said method comprises the addition of 0.5 to 4 % by weight, based on the total weight of the personal care composition, of at least one compound of formula I and at least one compound of Formula II. Both N-acyl L-prolines (Formula I) and N-acyl L-glutamines (Formula II) are useful personal care actives based on their antimicrobial profiles. N-acyl L-prolines are known for their skin benefits and are well-established in the personal care industry (Table 1, Example 4). N-acyl L-glutamines also exhibit antimicrobial properties. Based on their decent antimicrobial properties, N-acyl L-glutamines seem to have potential to be ‘skin and hair actives’ (Table 2a, Example 7). Amino acid residues as well as alkyl chains (length and saturation/unsaturation) of acyl residues of N-acyl L-amino acids can vary. This allows immense scope for creating efficacious synergistic combination of members of N-acyl L-proline family and members of N-acyl L-glutamine family. Table 2b reveals the minimum inhibitory concentrations of one such equimolar combination of N-cocoyl L-proline and N-capryloyl L-glutamine against a host of microbes connected with personal care at skin pH of 5.5. The MIC numbers of the combination seem to result the arithmetic average of the antimicrobial activities of the two individual antimicrobials. However, for effective protection of personal care products with pH range of 3.0 to 6.0, combinations with certain ratios are necessary. This is concluded by conducting preservation efficacy test (Evaluation protocol by Personal Care Product Council, Washington DC, USA; uses Gram positive/Gram negative bacteria, yeast and mold) of a cleanser based on ‘sulphate-free’ surfactants at three different pHs.
The scope of combinations of N-cocoyl L-proline and N-capryloyl L-glutamine is mapped by changing the ratios using a typical skin cleanser (body wash, Table 3a, Example 10) at three different pHs, 3.0, 5.5 and 6.0 using total N-acyl L-amino acids at maximum 1.5 %. It is observed that in the ‘sulphate-free’ surfactant-based formulation of Example 10, N-cocoyl L-proline works alone (clears the challenge test) if pH is highly acidic to moderate acidic at 1.5 % concentration, however it doesn’t pass the challenge test in formulation at pH 6.0 (entry 1, Table 3b). The entire range of pH from 3.0 to 6.0 gets adequately preserved when subjected to preservation efficacy test from the molar ratios of the two antimicrobials are varied from 2:1 to 1:2 (entries 3 to 7 of Table 3b). With the lower participation of strong antimicrobial partner (cocoyl proline) in entries 8 and 9 the formulation doesn’t pass the challenge test even at lower pHs.
In the above experiments the antimicrobials are used individually (Example 2 and Example 5) but with higher participation of N-acyl L-glutamines it is not possible to create fluid cold-dispersible combinations due to high melting solid nature of acyl glutamines. However, the working ratios (2:1 to 1:2) of Table 3a are amenable to fluid (cream/paste) with high concentrations of 70-75 % active and the rest being water as demonstrated in Example 8 and 9.
Thus combined, these molecules cover the shortcomings of individual acyl amino acids at different pHs of personal care formulations. The combinations of antimicrobials from the two families of acyl prolines and acyl glutamines are conveniently synthesized by one pot process to generate ‘cold dispersible’, concentrated yet fluid (creamy /pasty) and stable mixtures with water content of less than 30 % (Example 8 and Example 9). The one of synthesis of two families of N-acyl L-amino acids is accomplished by reacting fatty acid chlorides with corresponding L-amino acids at ambient conditions in the presence of a base in water. The Schotten-Baumann reactions are done sequentially and hence it allows any number of acyl amino acids to be synthesized in one pot. Yields are quantitative and the by-product is inorganic sodium chloride. The synthesis of the precursor fatty acid chloride is also done with a milder and safer chlorinating agent under ambient temperature using N-acyl amino-acid as catalyst (Step A of Example 1). The overall process of making blends of N-acyl L-amino acids of Examples 8 and 9 meet all the principles ‘Green’ chemistry and are used in preservation of water based formulation of Example 12 to Example 17.
Suitably the preservative system of present invention comprises the combination of N-acyl proline and N-acyl L-glutamine. Suitably the preservative system comprises combination of N-capryloyl L-proline and N-capryloyl L-glutamine, combination of N-nonyl L-proline and N-capryloyl L-glutamine, combination of N-decyl L-proline and N-capryloyl L-glutamine, combination of N- undecanoyl L-proline and N-capryloyl L-glutamine, combination of N-lauryl L-proline and N-capryloyl L-glutamine, combination of N-cocoyl L-proline and N-capryloyl L-glutamine, combination of N-myristoyl L-proline and N-capryloyl L-glutamine, combination of N-palmitoyl L-proline and N-capryloyl L-glutamine, combination of N-palmitoleoyl L-proline and N-capryloyl L-glutamine, combination of N-stearoyl L-proline and N-capryloyl L-glutamine, combination of N-oleoyl L-proline and N-capryloyl L-glutamine, combination of N-linoleoyl L-proline and N-capryloyl L-glutamine, combination of N-linonenoyl L-proline and N-capryloyl L-glutamine, combination of N-capryloyl L-proline and N-undecylenoyl L-glutamine, combination of N-nonyl L-proline and N- undecylenoyl L-glutamine, combination of N-decyl L-proline and N- undecylenoyl L-glutamine, combination of N- Undecanoyl L-proline and N- undecylenoyl L-glutamine, combination of N-lauryl L-proline and N- undecylenoyl L-glutamine, combination of N-cocoyl L-proline and N- undecylenoyl L-glutamine, combination of N-myristoyl L-proline and N- undecylenoyl L-glutamine, combination of N-palmitoyl L-proline and N- undecylenoyl L-glutamine, combination of N-palmitoleoyl L-proline and N- undecylenoyl L-glutamine, combination of N-stearoyl L-proline and N- undecylenoyl L-glutamine, combination of N-oleoyl L-proline and N- undecylenoyl L-glutamine, combination of N-linoleoyl L-proline and N- undecylenoyl L-glutamine, combination of N-linonenoyl L-proline and N- undecylenoyl L-glutamine.
More preferably the preservative system of present invention comprises 70% to 75% by weight of the preservative system, the combination of N-acyl L-proline and N-acyl L-glutamine.
Suitably the preservative system of present invention comprises the combination of N-acyl L-proline and N-acyl L-glutamine at the weight ratio of between 2:1 and 1:2.
Preferably the preservative of present invention comprises water from about 20 to 30 % by weight of preservative system.
Personal care ingredients:
The personal care ingredients used in the personal care composition of present invention are selected from anionic surfactants, amphoteric surfactants, non-ionic surfactants, cationic surfactants, pearlisers, opacifiers, emollients, anti-inflammatory, 100 % bio-renewable anti-microbial actives, UV-absorbers, UV-blockers, synthetic and/or bio-based polymeric conditioners, silicones, gums, rheology modifiers, polymeric rheology modifiers, film formers, vitamins, protein derivatives, anti-acne agents, anti-dandruff agents, moisturizers, humectants, emulsifiers, chelating agents, anti-oxidants, skin care active and hair active.
Suitable anionic surfactants are selected from but not limited to one or more soaps, salts of sulphuric ester or sulfates, salts of sulfonic acids or sulfonates, salts of phosphoric esters or phosphates, amino acids based anionic surfactants, isethionate based anionic surfactants, sarcosinate based amino acids surfactants, taurate based anionic surfactants. Other anionic surfactants generally known in art can also be the part of the anionic surfactants of present invention.
Suitable amphoteric surfactants are selected from but not limited to one ore more alkylamidopropylamine N-oxide, alkyldimethylamine N-oxide, alkylbetaine and alkylamidopropylbetaine. Cocamidopropyl betaine, cocoamphoacetate and cocoamphodiacetate are commonly known in art. Other amphoteric surfactants generally known in art can also be the part of the amphoteric surfactants of present invention.
Non-ionic surfactants are selected from but not limited to one or more alcohol ethoxylates, alkanolamides, surfactants derived from carbohydrates, ethoxylated alkanolamides, ethoxylated long chain amines, ethylene oxide/propylene oxide co-polymers, fatty acid ethoxylates, polyglyceryl ester and their ethoxylates, sorbitan derivatives, alkyl amines, alkyl imidazolines, ethoxylated oils and fats, alkyl phenol ethoxylates. Other non-ionic surfactants generally known in art can also be the part of the non-ionic surfactants of present invention.
Cationic surfactants are selected from but not limited to one or more quaternary ammonium derivatives, amines and imidazoline salts. Other cationic surfactants generally known in art can also be the part of the cationic surfactants of present invention.
Pearliser and opacifiers are selected from but not limited to glycol monostearate or etheylene glycol monostearate, ethylene glycol distearate, propylene glycol disterate.
Emollients are selected from but not limited to octyldodecyl myristate, ehtylhexyl stearate, alkyl benzoates, Caprylic/Capric Triglyceride, cocoyl ester of Caprylic/Capric.
Bio-renewable anti-microbial actives are selected from capryoyl glycine, undecylenoyl glycine, phenoxyethanol, glyceryl monoundecylenate, dehydroacetic acid and salts, benzoic acid and salts, sorbic acid and salts.
UV absorbers and UV blockers are selected from Oxybenzone, avobenzone, Homosalate, Octinoxate, octylmethoxy cinnamates, Galaxy Sunbeat, GalHuesheild, and other UV absorbers that are commonly known in the art.
Synthetic and/or bio-based polymeric conditioners are selected from Polyquaternariums, cellulose derivatives, acrylate polymers, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride.
Silicones are selected from dimethicone, methicone, cyclohexasiloxane, siloxane and their respective derivatives.
Gums are selected from Xanthan gum, Loctus bean gum, guar gum, arabic gum, cellulose derivatives.
Rheology modifiers and polymeric rheology modifiers are selected from cellulose derivatives and other synthetic polymers that are capable of modifying viscosity.
Vitamins are selected from those of synthetic origin.
Protein derivatives are selected from the various proteins are obtained from natural or synthetic sources and their derivatives or protein hydrolysate.
Anti-acne agents are selected from salicylic acid, Retinoids, benzoyl peroxide, antibiotics and mixture thereof.
Anti-dandruff agents are selected from piroctone olamine, ketoconazole (Sebizole), zinc pyrithione, and selenium disulfide, capryloyl glycine and mixture thereof.
Chelating agents are Disodium EDTA, Tetrasodium EDTA, Etidronic Acid, Oxalic Acid and derivatives, Phytic Acid.
Anti-oxidants are selected from glutathione, Ascorbic acid, Lipoic acid, Uric acid, Carotenes, vitamin E, coenzyme Q.
Skin and hair care actives are selected from plant extracts, molecules of synthetic origin and like that are capable to improve the skin appearance for example of face, or to reduce wrinkles, and lines of face or suitable anti-aging molecules.
Prebiotic property of the combinations of N-Acyl L-prolines and N-acyl L-glutamines towards skin-microbiota:
Staphylococcus epidermidis and Staphylococcus aureus are part of skin microbiota. In natural environment Staphylococcus epidermidis is a symbiont and prevents colonization of highly infectious Staphylococcus aureus which is highly contagious and causes serious diseases. Staphylococcus aureus spreads by direct contact with an infected person, by using a contaminated object or by inhaling infected droplets dispersed by sneezing or coughing. Skin infections from Staphylococcus aureus are common, and the bacteria can spread through the blood stream and infect distant organs. Skin infections may cause blisters, abscesses (furuncles), redness and swelling (Cellulitis) in the infected area. Staphylococcus aureus infects hair roots (Folliculitis) by developing a small pimple at the base of the hair. It also causes fluid filled blisters (Impetigo) on skin that burst. Staphylococcus aureus also causes Mastitis of breast after 1 to 4 weeks of the delivery, which may include cellulitis and abscesses. The area around the nipple becomes red and painful. Abscesses often release large numbers of bacteria into the mother’s milk. The bacteria may then infect the nursing infant. In children, S. aureus causes epidermal necrolysis that results in peeling of skin. Toxins of S. aureus also result in food poisoning and ‘toxic shock syndrome’. Some species are also known to release coagulase enzyme that results in blood clotting. Once it enters blood then it results in osteomyelitis or pneumonia. Also, many strains that originate from hospital or from hospital staff are antibiotic resistant and hence any antimicrobial that has decent activity against Staphylococcus aureus is welcome especially for topical application where resident Staphylococcus aureus can be the cause for not only skin/hair infections but other systemic infections and subsequent human to human spreading of the infections.
Thus, Staphylococcus aureus of skin microbiota is pathogenic and in contrast to this, the Staphylococcus epidermidis is commensal and involved in many host benefitting actions. It prevents colonization of S. aureus by secreting serine protease that destroys the biofilm of S. aureus (Iwase et al.; Nature, Letters Vol 465, p 246-250 (2010)). Modulins produced by S. epidermidis, along with host produced AMPs, selectively inhibit pathogens like S. aureus and Streptococcus. S. epidermidis is also involved in immunity pathway signaling and in anti-inflammatory activity by secreting lipoteichoic acid. (E. A. Grice and J. A. Segre, Nature Reviews: Microbiology, Volume 9, 243-253 (2011)).
Thus, the work by the scientific community over a decade (Julia Segre et al.; Nature Reviews, vol 116, p143-155, (2018)), has established that S. epidermidis is one of the major commensals that prevents colonization of pathogenic Staphylococcus aureus by three distinct pathways, 1) by destroying the biofilm of S. aureus by secreting serine protease glutamyl endopeptidase, 2) by signaling the immune cells and subsequently generating antimicrobial peptides, and 3) by generating the antibiotics, lantibiotics, that work synergistically with Cathilicidine.
It is also important to note that the biofilm forming property of infectious Staphylococcus aureus is supported by Propionibacterium acnes that produces coproporphyrin III to promote aggregation of Staphylococcus aureus (Julia Segre et al. Nature Reviews, vol 116, p143-155, (2018)). The antimicrobials combination of present invention shows a good activity against Cutibacterium acnes (Table 2b), and this indirectly helps prevent formation of biofilms by Staphylococcus aureus. Thus, the prebiotic combination of N-acyl L-proline and N-acyl L-glutamines of the present invention are capable of generating a prebiotic situation on the skin by creating higher population of commensal S. epidermidis which in turn will suppress pathogenic S. aureus by way of generating antimicrobial peptides as well as by generating film destroying enzyme and by biosynthesizing antibiotics. Thus, the combination of N-cocoyl L-proline and N-capryloyl L-glutamine of the present invention has huge potential to create a prebiotic effect on skin via personal care formulations, particularly via ‘leave-on’ applications (Example 11). Thus, the synergistic combination of antimicrobial preservatives of the present invention is a bio-degradable, bio-sourced, and prebiotic system for skin with other additional benefits derived from it like anti-dandruff, anti-acne and anti-malodor activity.
Preservation of personal care compositions with blends/combination of N-acyl L-prolines and N-acyl L-glutamines.
The personal care compositions of the present invention (Examples 12 to 21) are preserved with blends/combination of N-acyl L-glutamine and N-acyl L-proline at 1.0 to 2.0 % w/w of the total composition.
For evaluation of preservation efficacy, two combinations of N-acyl L-proline and N-acyl L-glutamine are prepared by a one pot process wherein two different N-acyl amino acids, sequentially, as described in Examples 8 and 9. These combinations have about 70 % mixture of N-acyl L-amino acids and the rest is water. At room temperature they form thin pastes/creams that are flowable or scoopable. These are easily cold-dispersible when added to water-based formulations. Example 8 is a combination of N-capryloyl L-glutamine and N-cocoyl L-proline in the molar ratio of 1:1 whereas Example 9 is combination of N-undecylenoyl L-glutamine and N-cocoyl L-proline in the molar ratio of 6:4. N-Acyl L-glutamines are solids with melting point above 120 ? and are not cold-dispersible, however, combinations of N-acyl L-glutamines and N-acyl L-prolines of Examples 8 and 9 are cold-dispersible. These are made by one pot process and isolated in 70-75 % active form (the rest is water).
The personal care compositions, mainly for cleansing of hair and skin (Examples 12-17), are preserved with the cold dispersible compositions of Example 8 or example 9. The complete homogeneity of these personal care formulations is obtained by mixing all ingredients at room temperature. Example 12 is a pearly shampoo with pearly sheen imparting ingredient 1,3 propane diol distearate whereas Example 13 is a ‘sulphate-free’ pearly body wash. Shampoo of Example 14 is preserved by the preservative blend of Example 9 (2.0 % of 70 % active) at pH 6.0. Extremely mild body wash of Example 15 with sophorolipids is preserved with 2.5 % of preservative blend of Example 8 (~70 % active) and pH of the body wash at 5.75. The combination of naturally derived N-capryloyl L-glutamine and N-cocoyl L-proline of Example 8 serves as preservative as well an effective anti-dandruff agent with very strong action against Malassezia restricta (Table 2a). For the same reason body washes with preservative systems of present patent application, are for derma hygiene due to their strong activity against the gram- positive bacteria (Table 2b).
While above shampoos and body washes are preserved at pH 5.5 and 6.0, a couple compositions with lower pH are preserved with antimicrobial blend of Example 8 in a facewash for acne at pH 4.5 (Example 16) and a feminine intimate hygiene wash at pH 3.5 (Example 17). In the face wash formulation, the composition of Example 8 with 4.5 pH comprising 2.0 % of preservative system, serves not only as preservative but also serves as an antiacne active (highly active again Cutibacterium acnes, Table 2b). Similarly, the composition of Example 8 in feminine hygiene wash of Example 17 at pH 3.5 serves as preservative for the composition as well as an active in controlling growth of Gardnerella vaginalis and Candida yeast (C. albicans and C. glabrata). All above ‘wash-off’ formulations passed the preservation efficacy test and shampoo formulations passed the test after rechallenging after 15 days at room temperature.
Composition of Example 8 is deployed in preservation of o/w cream (Example 18) and via this formulation, in addition to soothing and moisturizing effects, the disturbed microbiota (dysbiosis due to a skin condition) can be restored by the prebiotic effect, towards the skin, of N-capryloyl L-glutamine and N-cocoyl L-proline via their action on pathogenic Staphylococcus aureus (and Pseudomonas aeruginosa and Cutibacterium acnes, Table 4) and gentle action against symbiont, commensal Staphylococcus epidermidis (detailed experiment in Example 11).
Cold-processable preservative compositions of present invention are useful in cleansing bars (for both skin and hair, Example 21) as in addition to preservation it offers all the skin/scalp and hair hygiene benefits.
Water-less shower formulation (Example 20) also another gentle skin cleanser wherein the individual N-capryloyl L-glutamine (Example 5) and N-cocoyl L-proline (Example 2), oil-soluble at room temperature, serve as derma cleanser as well prebiotic towards skin microbiota.
Similarly, deodorant stick formulation (Example 19) is made with N-capryloyl L-glutamine (Example 5) and N-cocoyl L-proline (Example 2) to exploit extremely efficient action against the gram-positive bacteria of human axillary vault (Table 3).
The green (bio-resourced and biodegradable) antimicrobial preservation systems of present invention can be used with other similar ‘green’ antimicrobials or with petrochemical based but relatively non-toxic antimicrobials like capryloyl glycine, undecylenoyl glycine, benzoic acid or dehydroacetic acid (US 2023/0310292A1, Oct 5, 2023).
Advantages of the Invention
1) Bio-renewable feedstocks, green process of manufacture
Both antimicrobials of the present invention, N-acyl L-prolines (Formula I, RCO = C8 to C18) and N-acyl L-glutamines (Formula II, RCO = capryloyl and undecylenoyl), are derived from ‘bio-renewable sources’, namely, bio-renewable fatty acids (C8 to C18, saturated and unsaturated fatty acid), L-proline, and L-glutamine. Saturated or unsaturated fatty acids are derived from bio-renewable sources. Amino acid L-proline is manufactured from L-glutamic acid which is derived from fermentation of glucose. Similarly, L-Glutamine is industrially produced by fermentation of sugar using Brevibacterium flavum.
The syntheses of both N-acyl L-proline and N-acyl L-glutamine meet all twelve principles of ‘Green’ chemistry while producing individual N-acyl amino acid or producing members of both families together in one pot (Example 8 and 9). The processes of manufacturing don’t involve any hazardous reagents or dangerous reaction conditions, nor do they produce any wastage that needs to be disposed of. The yields of the processes are quantitative and there is no wastage at any stage.

2) Cold processable compositions of synergistic ‘green’ antimicrobials
Cold processable (easy to use) and stable compositions of the present invention are deployed in personal care products (Examples 12 to 17).
3) Biodegradable with 100 % RCI (renewable carbon index)
The combination of these two families of antimicrobials in 2:1 to 1:2 molar ratio results in naturality index of 100 %. Both N-acyl L-prolines (Formula I) and N-acyl L-glutamines (Formula II) are biodegradable and degraded products are harmless to the environment. The degraded products of fatty acids, L-proline and L-glutamine are well-metabolized by living systems.
4) Synergistic combination results in a broad spectrum of antimicrobial activity over a broad range of pH
The combination of N-acyl L-prolines and N-Acyl L-glutamines of the present invention is effective over a wide range of pH (3.0 to 6.0) that covers all personal care applications.
5) Prebiotic towards skin microbiota
The synergistic combination N-acyl L-prolines and N-acyl L-glutamines of the present invention is active against infectious Staphylococcus aureus and gentle on commensal Staphylococcus epidermidis. This synergistic combination helps commensal Staphylococcus epidermidis prevent the colonization of infectious Staphylococcus aureus (Example 11). The experiment explained in this Example 11 shows that when commensal S. epidermidis and pathogenic S. aureus are exposed in the presence of each other to the antimicrobial preservatives systems the present invention and then it is observed that the count for pathogenic bacterium is reduced by 2 log whereas the commensal is virtually unaffected. The protocol in terms of contact time etc. and the identification and distinction of colonies for counting is described in Example 11. Thus, the antimicrobial preservative combinations of the present invention show a very remarkable prebiotic effect towards skin microbiota.
6) Several personal care benefits in addition to preservation
The antimicrobial combinations of the present invention with N-acyl L-glutamines and N-acyl L-prolines offer several benefits in addition to preservation. They exhibit very good activity against microbes that are involved in proliferation of dandruff and acne, generation of mal-odor and feminine intimate hygiene. The preservative systems of the present invention comprising N-acyl L-proline and N-acyl L-glutamine, are linear fatty acid type molecules that blend well with skin lipids. The antimicrobial activity exhibited by these combinations is useful for preventing hyperproliferation (Cutibacterium acnes, Malassezia, and Candida) of skin microbiota. Thus, these serve as ‘Derma purifiers’ overall contributing to skin and scalp hygiene via both ‘rinse-off’ and ‘leave-on’ personal care products.
The preservative systems of the present invention, comprising N-acyl L-prolines and N-capryloyl L-glutamines, are active against the microbiota of axillary vault that mainly comprises of Staphylococci and Corynebacteria (Table 2b). Thus, in addition to being useful as a preservative system of personal care composition, it can control the microbes like Staphylococcus hominis, Staphylococcus epidermidis and Corynebacteria xerosis that degrade secretions of apocrine glands (sweat) to generate malodorous molecules.
7) Effective sustainable alternative to current toxic anti-microbials
The personal care compositions of the present invention comprising N-acyl L-prolines and N-acyl L-glutamines, are effective preservative alternative to the toxic (endocrine systems disruptors or neurotoxic to humans and ecology) antimicrobials that continue to destroy our planet.
8) Free from any potential toxic functionality
The components of the preservation systems, N-acyl L-prolines and N-acyl L-glutamines do not contain any inherent structural features such as phenolic moiety, halogens or any formaldehyde releasing functionality etc. that would be expected to be cytotoxic in future after prolonged usage.
9) Compatible with other personal care ingredients
The components of the present preservation system, N-acyl L-prolines and N-acyl L-glutamines of the present invention are compatible with all commonly used personal care ingredients.
10) Natural alternative to current toxic and fossil fuel based antimicrobial preservatives
As explained in the background that the arsenal of effective yet non-toxic antimicrobials has shrunk significantly to a very few gentle molecules with ‘limited antimicrobial activity’ like phenoxy ethanol, benzyl alcohol, benzoic acid, sorbic acid, dehydroacetic acid etc. Though these are relatively far less toxic compared to halogen containing antimicrobials, the major disadvantage they suffer from is their origin. These are not made from bio-renewable raw materials. All are synthesized from fossil fuel/petrochemicals (phenol, ethylene oxide, toluene, diketene, malonic acid and trans-butenal etc.). In view of this fact, the addition of N-acyl L-proline and N-capryloyl L-glutamine to the current limited arsenal of gentle natural antimicrobials opens several options of synergistic combinations for combinatorial approach for preservation.
Examples:
The present invention is now described by way of non-limiting illustrative examples. The details of the invention provided in the following examples are given by way of illustration only and should not be construed to limit the scope of the present invention.
All solid surfactant compositions are water-soluble/water dispersible at room temperature that are procured from Galaxy Surfactants Ltd, Mumbai, India and are without any antimicrobial preservatives.
1) Galsoft GLI 21(F): This is a solid (tiny flakes) product with 85 % active (sod cocoyl glutamate and sod cocoyl isethionate (2:1) and 15 % sodium chloride.
2) Galfusion VSF (PEG 150 distearate and sod methyl lauroyl taurate (8:2). This is made by melting PEG 150 distearate
3) GalGreen Sparkle (cold dispersible pearlizer with viscosity of 2000 to 3000 cps, 1,3 propane diol distearate and APGs)
4) Galsoft TMLI 12: This is a liquid (aqueous solution) with 45 % solids content and comprised of sodium lauroyl methyl taurate and sodium methyl lauroyl isethionate in the ratio of 1:2 by weight.
5) GalGreen Sparkle: This a cold-processable pearlizer based on 1,3 propane diol distearate ( (US 9,833,395), lauryl glucoside, polyglyceryl -4 laurate and sodium lauroyl lactylate. It is without any antimicrobial preservative and its solids content is 35 % with viscosity of < 4000 cps at 25 ?.
6) Galfusion Gentle Care 151 is aqueous solution (40-45 solids) of sodium methyl lauroyl taurate, sodium methyl lauroyl isethionate and cocoamidopropyl betaine in the ratio (on 100 % solids basis by weight) of 10:20:15:: sod methyl lauroyl taurate : sod lauroyl methyl isethionate : CAPB.
7) Galsophorin is the bio-surfactant, (sophorolipids) is procured from Galaxy Surfactants Limited, Mumbai, India. It is made by fermentation process with oleic acid and glucose as the carbon sources using the yeast, Starmerella bombicola ATCC 22214. It is an aqueous solution (50 %) and free from any externally added antimicrobial preservatives.
All other aqueous surfactants are used from in-house production (Galaxy Surfactants Ltd, India) to ensure that these do not contain any antimicrobial preservative. Examples of such aqueous surfactants are Galaxy CAPB-SB (cocoamidopropyl betaine 35%), GalsoftEcoCare 185 (lauryl glucoside, 50 %), Galsoft SCT -40 (sodium cocoyl taurate, 40 % aqueous paste) and Galsoft NaLS (sodium lauroyl sarcosinate, 30 %) and Galsoft SLGL (sodium lauroyl glutamate, 30 %). Galsilk -7 is polyquaternium-7, an aqueous solution with 9 % solids content.
Galsoft SCI -85 (P) is (Sodium cocoyl isethionate (85 %) in powder form. Galsoft SLT (N) is sodium lauroyl methyl taurate is 90 % surfactant in the needle form.
The MICs (minimum inhibitory concentrations) of N-acyl L-prolines and N-acyl L-glutamines and their combinations against various microbes are determined as per PCPC (Personal Care Product Council, Washington, USA) protocol.
Example 1: Synthesis of N-lauroyl L-proline:
It is synthesized by modified procedures based on the chemistry principles described in US 11771643. For making fatty acyl chlorides, the corresponding fatty acids are reacted with oxalyl chloride under the catalysis of N-acyl L-proline.
Step A: Synthesis of lauroyl chloride from lauric acid.
Lauric acid (200g, 1.0gmol) is reacted with oxalyl chloride (133g,1.05gmol) by slow addition over period of 2 hours at 35 ? under nitrogen blanket and with a trap to absorb liberated carbon dioxide. This reaction is catalyzed by (0.6g) N-lauroyl proline which is mixed with molten lauric acid (48 ?) at the beginning of the reaction. Excess and the subsequent traces of acidity are removed by purging N2 through the reaction mass. Lauroyl chloride thus obtained is an almost colorless liquid with free lauric acid around 1 %. This lauroyl chloride is used for the second step of synthesis of N-lauroyl L-proline.
Step B: Synthesis of N-lauroyl L-proline
To a stirred mixture of L-proline (118.5 g, 1.03 gmol) solution in water (410 mL) at 15-25 oC under nitrogen, lauroyl chloride (223 g, 1.0 gmol) and 48 % aqueous solution of sodium hydroxide (170 g, 2.03 gmol) are added simultaneously over the period of 3 hrs. while maintaining pH of the reaction mass in the range of 10.5-11.5. The reaction mixture is further stirred for one hour at the 25-30 ? temperature. Sodium N-lauroyl L-prolinate, thus formed, is a clear liquid: Quantity: 920 g; Solids content: 42.30 %; NaCl: 6.25%.
The clear aqueous solution of sodium N-lauroyl L-prolinate (920 g) thus obtained is acidified with hydrochloric acid solution (110 g, 1.05 gmol) in the second step at room temperature and the pH of the reaction mass is adjusted in the range of 1.5 to 2.5. The aqueous layer is then removed and the upper organic layer of N-lauroyl proline is washed (100 mL) of fresh water to remove traces of mineral acidity in the organic layer. The washed organic phase is further dried using rotary evaporation at room temperature under vacuum to afford acyl proline as low viscous pale-yellow colored liquid (Yield: 295 g, 99%. %; Acid Value: 188, Moisture Content: 0.25 %). Optical rotation for N-lauroyl L-proline : - 2.7º ( 20? , 5 % in methanol, 589 nm)
Example 2: Synthesis of N-cocoyl L-proline
In an analogous manner cocoyl chloride is prepared from coco fatty acid and oxalyl chloride catalyzed by N-cocoyl L-proline. Cocoyl chloride thus obtained is used the synthesis of N-cocoyl L-proline as described below. The composition of saturated coco fatty is; C8 5.0 %, C10 5%, C12 63%, C14 21%, C16 5%, C18 0.5 %,
To a stirred mixture of L-proline (118.5 g, 1.03 gmol,) solution in water (410 mL) at 15-25 oC under nitrogen, cocoyl chloride (223 g, 1.0 gmol) and 48 % aqueous solution of sodium hydroxide (171 g, 2.03 gmol) are added simultaneously over the period of 3 hrs. while maintaining pH of the reaction mass in the range of 10.5-11.5. The reaction mixture is further stirred for one hour at the 25-30 ? temperature. Sodium N-cocoyl L-prolinate, thus formed, is a clear liquid: Quantity: 922 g; Solids content: 42.20 %; NaCl: 6.29%.
The clear aqueous solution of sodium N- cocoyl L-prolinate (922 g) thus obtained is acidified with hydrochloric acid solution (110 g, 1.05 gmol) in the second step at room temperature and the pH of the reaction mass is adjusted in the range of 1.5 to 2.5. The aqueous layer is then removed and the upper organic layer of N-lauroyl proline is washed (100 mL) of fresh water to remove traces of mineral acidity in the organic layer. The washed organic phase is further dried using rotary evaporation at room temperature under vacuum to afford acyl proline as low viscous pale-yellow colored liquid (Yield: 293 g, 99%. %; Acid Value: 189; Moisture Content: 0.18 %). Optical rotation for N-lauroyl L-proline : - 2.6º (20 ? , 5 % in methanol, 589 nm).
Example 3: Synthesis of N-capryloyl L-proline:
To a stirred mixture of L-proline (118.5 g, 1.03 gmol,) solution in water (340 mL) at 15-25 oC under nitrogen, capryloyl chloride (165 g, 1.0 gmol) and 48 % aqueous solution of sodium hydroxide (171g, 2.03 gmol) are added simultaneously over the period of 3 hrs. while maintaining pH of the reaction mass in the range of 10.5-11.5. The reaction mixture is further stirred for one hour at the 25-30 ? temperature. Sodium N-capryloyl L-prolinate, thus formed, is a clear liquid: Quantity: 792 g; Solids content: 42.40 %; NaCl: 7.34%.
The clear aqueous solution of sodium N-capryloyl L-prolinate (792 g) thus obtained is acidified with hydrochloric acid solution (110 g, 1.05 gmol) in the second step at room temperature and the pH of the reaction mass is adjusted in the range of 1.5 to 2.5. The aqueous layer is then removed and the upper organic layer of N-lauroyl proline is washed (100 mL) of fresh water to remove traces of mineral acidity in the organic layer. The washed organic phase is further dried using rotary evaporation at room temperature under vacuum to afford acyl proline as low viscous pale-yellow colored liquid (Yield: 238 g, 99%. %; Acid Value: 231.25; Moisture Content: 0.27 %). Optical rotation for N-lauroyl L-proline: - 3.2º (20?, 5 % in methanol, 589 nm).
Example 4: Minimum inhibitory concentrations of N-acyl L-prolines (pH 5.5)
Table 1: Minimum inhibitory concentration of N-acyl L-prolines
MIC (%)
N-Capryloyl L-proline MIC (%)
N-Cocoyl L-proline MIC (%)
N-Lauroyl L-proline
Gram positive
Staphylococcus aureus ATCC 6538 0.3 0.01 0.01
Cutibacterium acnes ATCC 6919 0.4 0.3 0.1
Staphylococcus epidermidis NCIM 2493 1 1 1
Staphylococcus hominis MTCC 8980 0.4 0.01 0.01
Micrococcus luteus NCIM 2103 0.5 0.01 0.03
Corynebacterium xerosis ATCC 373 0.5 0.08 0.05
Gram variable
Gardnerella vaginalis ATCC 14018 0.4 0.01 0.01
Gram negative
Escherichia coli ATCC 8739 0.5 0.7 1
Pseudomonas aeruginosa ATCC 15442 0.5 0.7 1
Burkholderia cepacia ATCC 25416 1 0.8 1
Yeast
Candida albicans ATCC 10231 0.4 0.5 0.2
Candida glabrata ATCC 66032 0.4 0.5 0.1
Malassezia furfur ATCC 14521 0.4 0.1 0.06
Malassezia restricta ATCC MYA 4611 0.3 0.02 0.02
Mold
Aspergillus niger ATCC 16404 1 1 1

Example 5: Synthesis of N-capryloyl L-glutamine
To a stirred solution of L-glutamine under nitrogen blanket (42 g, 0.28 mol) in water (352 ml) with pH of 10.5 adjusted with NaOH (48%) capryloyl chloride is added (44.5 g, 0.27 mol) while maintaining the pH of the reaction mass between 10.5 to 11.5 with simultaneous addition of sodium hydroxide (48 % solution, 46 g, 0.54 mol) solution at 20 - 30°C. After completion of the addition of capryloyl chloride, the reaction mass is further stirred for 2 h. The reaction mass is acidified with concentrated hydrochloride acid to pH 1.5 and the precipitated solid is filtered and washed with water to remove the mineral acidity. Drying wet cake yielded N-capryloyl L-glutamine as white solid (74 g, 99 % yield).
FTIR: (as such) 1732, 1671, 1642, 1621, 3437 and 3355, 3309 cm-1
Melting point: 120 - 122°C, specific optical rotation: -3.463°, Concentration = 4% in methanol at 20°C and 589 nm.
1H NMR (500 MHz, DMSO-d6) d 12.48 (s, 1H, -COOH), 8.04 (d, J = 7.7 Hz, 1H, -CONHCH-), 7.28 (s, 1H, -CONH2), 6.77 (s, 1H, -CONH2), 4.13 (td, J = 8.5, 5.3 Hz, 1H, -NH-CH-COO-), 2.11 (q, J = 7.0 Hz, 4H), 1.92 (m, 1H), 1.72 (m, 1H), 1.50-1.46 (m, 2H), 1.28 – 1.24 (m, 8H), 0.85 (t, J = 6.8 Hz, 3H).
13C NMR (100 MHz, CDCl3) d: 174.73, 173.32, 172.99, 51.33, 35.66, 31.28, 28.53, 27.36, 24.95, 21.90, 13.44.
Example 6 : Synthesis of N-undecylenoyl L-glutamine
To a stirred solution of L-glutamine (153.5 g, 1.05 gmol) in water (350 ml) with pH of 10.5adjusted with NaOH (48%) undecylenoyl chloride is added (204 g, 1.0 gmol) while maintaining the pH of the reaction mass between 10.5 to 11.5 with simultaneous addition of sodium hydroxide (48 % solution, 170 g, 2.05 gmol) solution at 15 – 30 °C. After completion of the addition of undecylenoyl chloride, the reaction mass is further stirred for 2 h. The reaction mass is acidified with concentrated hydrochloride acid to pH 1.5 and the precipitated solid is filtered and washed with water to remove the mineral acidity. Drying wet cake yielded N-undecylenoyl L-glutamine as white solid (305 g, 98.% yield).
FTIR: (as such) 1732, 1671, 1642, 1621, 3437 and 3355, 3309 cm-1
Melting point: 117-121°C, Specific optical rotation: -2.814°, Concentration = 4% in methanol at 20°C and 589 nm.
1H NMR (500 MHz, DMSO-d6) d 12.48 (s, 1H, -COOH), 8.05 (d, J = 7.7 Hz, 1H, -CONHCH-), 7.29 (s, 1H, -CONH2), 6.77 (s, 1H, -CONH2), 5.78 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H, -CH2=CH-CH2-), 4.99 (dd, J = 17.1, 1.4 Hz, 1H, -HtransCH=CH-CH2-), 4.92 (d, J = 10.2 Hz, 1H), 4.13 (td, J = 8.5, 5.2 Hz, 1H, -NH-CH-COO-), 2.10 (q, J = 7.0 Hz, 4H), 2.00 (q, J = 7.0 Hz, 2H), 1.92 (m, 1H), 1.72 (m, 1H), 1.52 – 1.42 (m, 2H, alkyl chain), 1.38 – 1.29 (m, 2H, alkyl chain), 1.24 (s, 8H, alkyl chain).
13C NMR (125 MHz, DMSO-d6) d 174.0 (-COOH), 173.9 (-CONH-), 172.7 (-CONH2-), 139.2 (H2C=CH-), 115.0 (H2C=CH-), 51.9 (NH-CH-COOH), 35.5, 33.6, 31.8, 29.2, 29.0, 28.9, 28.7, 27.3, 25.6, 24.9.
Example 7: Minimum inhibitory concentrations of N-acyl L-glutamines
Table 2a: Minimum inhibitory concentrations of N-acyl glutamines.
Microorganisms N-Capryloyl L-glutamine N-Undecylenoyl L-glutamine
MIC (in %) MIC (in %)
Staphylococcus aureus ATCC 6538 0.3 0.2
Cutibacterium acnes ATCC 6919 0.2 0.2
Staphylococcus epidermidis NCIM 2493 0.9 0.6
Staphylococcus hominis MTCC 8980 0.6 0.2
Micrococcus luteus NCIM 2103 0.5 0.1
Corynebacterium xerosis ATCC 373 0.5 0.6
Gardnerella vaginalis ATCC 14018 0.6 0.2
Escherichia coli ATCC 8739 0.7 1
Pseudomonas aeruginosa ATCC 15442 0.7 1
Burkholderia cepacia ATCC 25416 1 1
Candida albicans ATCC 10231 0.6 0.6
Candida glabrata ATCC 66032 0.6 0.4
Malassezia furfur ATCC 14521 0.3 0.2
Malassezia restricta ATCC MYA 4611 0.4 <0.1
Aspergillus niger ATCC 16404 1 1

Example 8: Preparation of a blend of N-cocoyl L-proline and N-capryloyl L-glutamine in 1:1 ratio by weight and minimum inhibitory concentration
To a stirred solution of L-glutamine (76 g, 0.515 gmol) in water (319 ml) with pH of 10.5 adjusted with NaOH (48 %) capryloyl chloride is added (81 g, 0.5 gmol) while maintaining the pH of the reaction mass between 10.5 to 11.5 with simultaneous addition of sodium hydroxide (48 % solution, 86 g, 1.03 gmol) solution at 15 – 30 °C. After completion of the addition of capryloyl chloride, the reaction mass is further stirred for 2 h. To this stirred mass additional water and L-proline (59.5 g,0.515 gmol) are added. To this stirred mass, cocoyl chloride (110 g, 0.5 gmol) and caustic lye (48 %, 86 g , 1.03 gmol) are added simultaneously maintaining the temperature between 15 to 30 ? and the pH of the reaction mass between 10.5 to 11.5.
The reaction mass is acidified with concentrated hydrochloride acid to pH 1.5 and temperature of the mass is raised to 60 ? and the mixture of N-cocoyl L-proline and N-capryloyl L-glutamine is phase separated from the acidic water. The mixture of N-acyl amino acids is further washed with water (110 g) while maintaining the temperature at 80-90 ?. It is further phase separated from the water layer as pale-yellow colored liquid which turned into a flowable cream after attaining ambient temperature (365 g). The flowable pale yellow colored pasty mass of two N -acyl amino acids with the analysis mentioned below is obtained. This is further used in preservation of several personal care compositions where water is permitted.
Water content: 25 %, Acid value :137, sodium chloride content: 0.9 %, pH of 1 % dispersion: 2.8. Active content of about 68 to 73%.
Table 2b: Minimum inhibition concentration of combination of N-acyl L proline and N-acyl L-glutamine – The preservative system of present invention
N-Capryloyl L-glutamine N-Cocoyl L-proline N-Cocoyl L-proline: N-capryloyl L-glutamine (1:1)
Staphylococcus aureus ATCC 6538 0.3 0.01 0.09
Cutibacterium acnes ATCC 6919 0.2 0.3 0.2
Staphylococcus epidermidis NCIM 2493 0.9 1 0.5
Staphylococcus hominis MTCC 8980 0.6 0.01 <0.1
Micrococcus luteus NCIM 2103 0.5 0.01 <0.1
Corynebacterium xerosis ATCC 373 0.5 0.08 0.3
Gardnerella vaginalis ATCC 14018 0.6 0.01 0.1
Gram negative
Escherichia coli ATCC 8739 0.7 0.7 0.7
Pseudomonas aeruginosa ATCC 15442 0.7 0.7 0.7
Burkholderia cepacia ATCC 25416 1 0.8 1
Candida albicans ATCC 10231 0.6 0.5 0.5
Candida glabrata ATCC 66032 0.6 0.5 0.6
Malassezia furfur ATCC 14521 0.3 0.1 0.2
Malassezia restricta ATCC MYA 4611 0.4 0.02 0.1
Aspergillus niger 1 1 1

Example 9: Preparation of a blend of N-Undecylenoyl L-glutamine and N-cocoyl L-proline in 3:2 ratio by weight.
To a stirred solution of L-glutamine (88.8 g, 0.60 gmol) in water (339.5ml) with pH of 10.5 adjusted with NaOH (48%) undecylenoyl chloride is added (119.5g, 0.59gmol) while maintaining the pH of the reaction mass between 10.5 to 11.5 with simultaneous addition of sodium hydroxide (48 % solution,100 g, 1.2 gmol) solution at 15 – 30 °C. After completion of the addition of capryloyl chloride, the reaction mass is further stirred for 2 h. To this stirred mass L-proline (47.5 g, 0.412 gmol) is added. This is followed by simultaneous addition of cocoyl chloride (89.5g, 0.41gmol) and caustic lye (48 % , 70 g ,0.84 gmol) maintaining the temperature between 15 to 30 ? and the pH of the reaction mass between 10.5 to 11.5.
The reaction mass is acidified with concentrated hydrochloride acid to pH 1.5 and temperature of the mass is raised to 60 ? and the mixture of N-cocoyl L-proline and N-undecylenoyl L-glutamine is phase separated from the acidic water. The mixture of N-acyl amino acids is further washed with water (110g) while maintaining the temperature at 60 80-90?. It is further phase separated from the water layer as pale-yellow colored liquid which turned into a cream after attaining ambient temperature (400g). The pasty/creamy mass of water and the two N -acyl amino acids is further used in preservation of several personal care compositions where water is permitted.
Analysis of this creamy mass: Water content 25.50 %, Acid value 157.2, sodium chloride content: 1.20 %, pH of 1 % dispersion: 2.90, Active content of about 68 to 73%.
Example 10: Preservation efficacy of N-cocoyl L-proline, N-capryloyl L-glutamine and their combinations in a cleansing surfactant composition.
Using a typical surfactant-based cleansing composition three sets are prepared, at pH, 3.5, 5.5 and 6.0. Antimicrobials individually and in combination are tested for preservation efficacy as per the PCPC protocol.
Table 3a: Cleansing formulations I, II and III with pH 3.5, 5.5 and 6.0 respectively
Ingredients (I)
w/w% (II)
w/w% (III)
w/w%
Distilled Water q.s. to 100% q.s. to 100% q.s. to 100%
Sodium lauroyl glutamate (Galsoft SLGL (30%) 18.0% 18.0% 18.0%
Sodium cocoyl glutamate and sodium cocoyl isethionate (2:1, flake) (Galsoft GLI 21 (F)) 7.0 % 7.0% 7.0%
Galaxy CAPB (35 % solids) 13.0% 13.0% 13.0%
Glycerin 2.0% 2.0% 2.0%
Antimicrobial (N-capryloyl L-glutamine or N-cocoyl L-proline or combination of the two) q.s q.s q.s
Citric acid (50% aq. sol) q.s. pH = 3.5 q.s. pH = 5.5 q.s. pH = 6.0

Method of preventing microbial contamination of a personal care composition as per present invention is demonstrated in below table. The personal care compositions prepared as per table 3a are preserved with the respective ratio of the preservative system of the present invention. The preservative system, prepared as per the respective ratio in below table 3b, is added to the formulation of table 3a and the personal care formulations are challenged for preservation efficacy, so as to prevent microbial contamination, as per PCPC protocol.
Table 3b: Preservation efficacy tests on Formulations at pH 3.5, 5.5 & 6.0 with N-cocoyl L-proline and N-capryloyl L-glutamine
Sr No. N-cocoyl L-proline %
Example 2 N-capryloyl L-glutamine %
Example 5 Ratio Challenge test
pH 3.5 Challenge test
pH 5.5 Challenge test
pH 6.0
1 1.5 % 0 % 100: 0.0 Passes Passes Fails
2 1.2 % 0.3 % 80:20 Passes Passes Fails
3 1.0 % 0.5 % 66:33 Passes Passes Passes
4 0.9 % 0.6 % 60:40 Passes Passes Passes
5 0.75 % 0.75 % 50:50 Passes Passes Passes
6 0.6 % 0.9 % 40:60 Passes Passes Passes
7 0.5 % 1.0 % 33:66 Passes Passes Passes
8 0.3 % 1.2 % 20:80 Passes Fails Fails
9 0 % 1.5 % 0.0 : 100 Passes Fails Fails

Example 11: Combination of N-cocoyl L-proline and N-capryloyl L-glutamine as a prebiotic preservative system
A combination of N-cocoyl L-proline and N-capryloyl L-glutamine (1:1) by weight is selected for establishing highly selective action against Staphylococcus aureus in the presence of Staphylococcus epidermidis. A prebiotic substance supports the good microbes of human microbiota that are also actively involved in what is called ‘natural’ or ‘innate’ immunity in protecting humans from pathogenic microbes. Both N-capryloyl L-glutamine and N-cocoyl L-proline do support commensal good bacteria Staphylococcus epidermidis by selectively going aggressively against the pathogenic Staphylococcus aureus which competes with S. epidermidis for both space and food.
The test strain suspensions of S. aureus and S. epidermidis are adjusted to a count of 104 CFU/ml.
For the test preparation, 500 µl of the product is mixed with 5.2 ml PBS (phosphate buffer saline) and 300 µl of TSB (Tryptic Soy Broth). Subsequently, of the respective 100 µl S. aureus and 500 µl S. epidermidis test strain suspensions are inoculated. A control batch containing 500 µl of PBS instead of the product is included. The tests and control are incubated in orbital shaker (120 rpm) for 4 hrs. for at 37°C. After incubation, the preparation (500 µl) is removed, centrifuged at 13,000 rpm and then the pellet is redissolved (resuspended) with 500 µl PBS. This suspension (500 µl) is added to TSB (4.5 ml) for serial dilution and CFU/ml is determined using Tryptic Soy Agar. The plates are incubated at 35°C for 48hrs. Golden yellow colonies of S. aureus are easily distinguishable from white cream-colored colonies of S. epidermidis on Tryptic Soy Agar (Fig1) . The results are tabulated below.
Table 4: Count of S. aureus and S. epidermidis in control and test in petri dish
S. aureus S. epidermidis
Control (cfu/ml) 7.2×104 4.3×107
Test (cfu/ml) 7.2×102 1.3×107
Log reduction 2 0.50

The count for Staphylococcus aureus in the ‘control’ is 7.2×104 whereas the count for S. epidermidis is 4.3×107. However, in the ‘test’ the S. aureus count is reduced completely and S. epidermidis count is almost unaltered and quite same as the ‘control’. In the presence of N-cocoyl L-proline and N-capryloyl L-glutamine, the count of pathogenic S. aureus is completely inhibited compared to commensal S. epidermidis. A log reduction of 2 is observed for S. aureus and 0.50 for S. epidermidis.
In another set of experiments with short incubation period of 15 mins, 1 log reduction for Staphylococcus aureus is seen and for Staphylococcus epidermidis there is virtually no reduction in the count (< 0.1). This experiment is done to estimate the possibility of prebiotic effect of the antimicrobial combo of the present invention in ‘rinse-off’ cleansers with short contact time with human skin.
Table 5: Count of S. aureus and S. epidermidis in control and test in petri dish with short incubation period of 15 minutes
S. aureus S. epidermidis
Control (cfu/ml) 3×104 1.5×107
Test (cfu/ml) 2.5×103 1.3×107
Log reduction 1.07 0.05

Example 12: Preparation of ‘sulphate-free’ shampoo and its preservation with blend of Example 8.
Ingredients Trade name/Supplier Weight %
Water - To make 100.0
Sodium lauroyl sarcosinate (30%) Galsoft NaLS 5.0
Sophorolipids (50%) GalSophorin 7.0
Sodium cocoyl taurate (40%) Galsoft SCT 40 15.0
Cocamidopropyl betaine (36%) Galaxy CAPB 10.0
Lauryl polyglucoside (50%) Galsoft EcoCare 195 10.0
Glycerin - 2
Sodium gluconate - 0.2
Tetrasodium glutamate diacetate Aquacid 2024 EX 0.2
Polyethylene glycol 150 distearate and sodium lauroyl taurate (80:20) Galfusion VSF 1.5
1,3-propane diol distearate and lauryl glucoside, polyglyceryl 4 luarate, polyglyceryl-4, and sodium lauroyl lactylate GalGreen Sparkle 3.0
N-Cocoyl L-proline & N-Capryloyl L-glutamine (70 % active) Example 8 2.0
Polyquaternium -7 (9 %) Galsilk-7 3.0

Procedure: To water (50 parts), all the ingredients are added sequentially, and gentle agitation is continued at room temperature till homogeneous mass is obtained. If necessary, pH is adjusted to 5.75 with either citric acid or sodium hydroxide. Balance amount of water is added to make up to 100. The resultant formulation has viscosity of 3850 cps at 25 °C. The composition described above passes the preservation efficacy test.
Example 13: Preparation of body-wash and its preservation with N-undecylenoyl L-glutamine and N-cocoyl L-proline
Ingredients Trade names/Supplier Weight %
Water - To make 100.0
Sodium cocoyl glutamate and sodium cocoyl isethionate (2:1) Galsoft GLI 21 (Flakes) 10.00
Lauryl polyglucoside (50%) Galsoft EcoCare 195 8.00
Cocamidopropyl betaine (36%) Galaxy CAPB 18.00
Tetrasodium glutamate diacetate Aquacid 2014 EX 0.30
Polyethylene glycol 150 distearate + Sodium lauroyl taurate (80:20) Galfusion VSF 0.75
N-undecylenoyl L-glutamine and N-cocoyl L-proline (70 %) Example 9 2.1
1,3 propanediol distearate, lauryl glucoside, polyglyceryl 4 laurate, polyglyceryl-4 and sodium lauroyl lactylate GalGreen Sparkle - 6.00
Procedure: To water (50 parts), all the ingredients are added sequentially, and gentle agitation is continued at room temperature till homogeneous mass is obtained. If necessary, pH is adjusted to 5.90 with either citric acid or sodium hydroxide. Balance amount of water is added to make up to 100. The resultant formulation has viscosity of 12500 cps at 25 °C. The composition described above passes the preservation efficacy test.
Example 14: Preparation of ‘sulphate-free’ shampoo and its preservation with N-undecylenoyl L-glutamine and N-cocoyl L-proline (3:2, Example 9).
Ingredients Trade name/Supplier Weight %
Water - To make 100.0
Sodium lauroyl sarcosinate (30%) Galsoft NaLS 15.0
Sophorolipids (50%) Galsophorin 7.0
Sodium lauroyl taurate (90 %, needle) Galsoft SLT -N 6.0
Cocamidopropyl betaine (36%) Galaxy CAPB 10.0
Lauryl polyglucoside (50%) Galsoft EcoCare 195 5.0
Glycerine - 2
Sodium gluconate 0.2
Tetrasodium glutamate diacetate Aquacid 2024 EX 0.2
Polyethylene glycol 150 distearate and sodium lauroyl taurate Galfusion VSF 3.0
1,3 propane diol distearate, alkyl poly glucosides, polyglyceryl 4 laurate, and sodium lauroyl lactylate GalGreen Sparkle 3.0
N-Undecylenoyl L-glutamine and N-cocoyl L-proline ( 70 %) Example 9 2.0
N-capryloyl L-proline Example 3 0.5
Polyquaternium -7 (9 %) Galsilk-7 3.0

Procedure: To water (50 parts), all the ingredients are added sequentially, and gentle agitation is continued at room temperature till homogeneous mass is obtained. If necessary, pH is adjusted to 6 with either citric acid or sodium hydroxide. Balance amount of water is added to make up to 100. The resultant formulation has viscosity of 5400 cps at 25 °C. The composition described above passes the preservation efficacy test.
Example 15: Preparation of ‘extra mild’ body-wash and its preservation with N-cocoyl L-proline & N-capryloyl L-glutamine (Example 8)
Ingredients Trade Name/Supplier Weight %
Water - To make 100.0
Sophorolipids (50%) Galsophorin 5.0
Sodium cocoyl glutamate and sodium cocoyl isethionate Galsoft GLI 21 (Flake) 12
Cocoamidopropyl betaine (36%) Galaxy CAPB 15
Tetra sodium glutamate diacetate Aquacid 2014 EX 0.2
N-cocoyl L-proline & N-capryloyl L-glutamine (70 %) Example 8 2.5
Polyethylene glycol 150 distearate and sodium lauroyl taurate Galfusion VSF 2.0
Citric acid - To pH 5.5 to 5.8
Procedure: In gently stirred water (60 parts) at room temperature, all the surfactants are added in sequence, namely, Galsophorin, Galsoft GLI 21 (flake) and cocoamidoproyl betaine. This is followed by the chelating agent and the antimicrobial preservative of this invention. Finally, Galfusion VSF is added and stirring is continued for additional two hours at ambient temperature till the mixture is homogeneous. pH is adjusted to 5.75. The resultant formulation has viscosity of 7900cp at 25°C. This composition passes the challenge test for preservation.
Example 16: Preparation of facewash and its preservation blend of Example 8
Ingredients Trade name/Supplier Weight %
Water - To make 100
Sodium lauroyl methyl taurate & Sodium lauroyl methyl isethionate, (40%, 2:1) Galsoft TLMI 12 15
Cocamidopropyl betaine (36%) Galaxy CAPB 21.7
Polyethylene glycol 150 distearate and sodium lauroyl taurate Galfusion VSF 1.0
Glycerine VVF 5.00
Tetrasodium glutamate diacetate Aquacid 2014 EX 0.2
N-cocoyl L-proline & N-capryloyl L-glutamine (70 % active) Example 8 2.0
Lactic acid solution (88% w/w solution) - To adjust pH to 4.5
Procedure: To water (65 parts), Galsoft TLMI 12 (15 parts) is added under slow agitation. This is followed by other ingredients and stirring is continued at room temperature till a homogeneous blend is obtained. pH is adjusted to 4.5 with lactic acid and balance amount of water is added under slow agitation at ambient temperature. The resultant formulation has viscosity of 2980 cps at 25 °C. This composition passes the preservation efficacy test.
Example 17: Preservation of intimate feminine hygiene wash with a combination of (Example 8) N-cocoyl L-proline and N-capryloyl L-glutamine.
Ingredients Trade Name/Supplier Weight %
Water - To make 100.0
Lauryl polyglucoside (50%) Galsoft EcoCare 195 10.00
Sodium lauroyl taurate and sodium lauroyl methyl isethionate (1:2 ratio), 40 % solution Galsoft TLMI:12 40.00
Cocamidopropyl betaine (36%) Galaxy CAPB 10.00
N-capryloyl L-glutamine and N-cocoyl L-proline (70 % active) Example 8 2.5
Tetrasodium glutamate diacetate Aquacid 2014 EX 0.2
Lactic acid solution (90 % w/w solution) Purac Hipure 90 To adjust pH 3.5

Procedure: All surfactants (first three) are added together and stirred gently at room temperature. To this stirred mass N-capryloyl L-glutamine and N-cocoyl L-proline (70 % active, Example 8) and sodium gluconate are added. After achieving a homogeneous solution, pH is adjusted to 3.8 with lactic acid. The resultant formulation has viscosity of 200 cp at 25 °C. This composition passes the challenge test.
Example 18: Preservation of a cream with the blend of Example 8 (N-cocoyl L-proline and N-capryloyl L-glutamine)
Ingredients Trade names/Supplier Name Weight %
Phase A
Water - To make 100.0
Glycerin - 4.0
Polyethyleneglycol-7-glyceryl cocoate Galaxy PEG-7 GC 2.0
Sodium gluconate - 0.2
Tetrasodium glutamate diacetate Aquacid 2014 EX 0.2
Phase B
Cetostearyl alcohol VVF LLC 7.0
Stearic acid Emery Oleochemicals 2.0
Glyceryl mono stearate Fine Organics 5.0
Isopropyl myristate - 2.5
N-Cocoyol L-proline & N-capryloyl L-glutamine ( 70 %) Example 8 2.2

Procedure: The contents of Phase A and Phase B are heated separately up to 75°C, with stirring. Phase B is then added to Phase A with constant stirring and homogenized (Silverson) for 30 minutes until emulsion is obtained. The cream is then cooled to room temperature and stirring is continued until uniform consistency is obtained. pH of the final formulation is adjusted to 5.5 with caustic lye and optional fragrance and color are blended uniformly. The resultant formulation has viscosity of 5000 cp at 25 °C. The composition passes the preservation efficacy test.
Example 19: Preservation of a deodorant stick (N-cocoyl L-proline and N-capryloyl L-glutamine)
Ingredients Trade Name/Supplier Weight %
Paraffin Light Liquid - 38.00
Bees Wax Arjun Beeswax 31.00
Paraffin Wax Golden Dyechem 11.00
Shea Butter Samala Udyog 11.00
Cetaryl alcohol VVF 06.00
N-Cocoyl L-proline Example 2 0.5
N-capryloyl L-glutamine Example 5 0.5
Vitamin E acetate Matrix Life science 0.9
Dimethicone Dow 1.5

Procedure: All ingredients are heated together to 75 to 80 °C and gently mixed till a homogeneous mass is formed. It is cooled to 60 °C under gentle stirring. The homogeneous mass is added to the cast and allowed to cool to room temperature.
Example 20: Preparation of water-less shower formulation containing dermapurifiers, N-cocoyl L-proline and N-undecylenoyl L-glutamine
Ingredients Trade Name/Supplier Weight %
Phase A
Potassium N-cocoyl L-prolinate (100%) - 16.00
Polyglyceryl 3-oleate Fine Organics 30.00
N-undecylenoyl L-glutamine Example 6 0.50
N-cocoyl L-proline Example 2 0.50
Phase B
Triglycerides AAK India 50.00
Glyceryl mono laurate Finester LG 9000 3
Vitamin E acetate Matrix life science 0.5

Phase A ingredients are mixed and stirred at room temperature until homogeneous. Phase B is separately made by mixing all ingredients at room temperature. Phase B is added to Phase A under stirring till the mixture acquires homogeneity.
Example 21: Preparation of anti-odor skin hygiene cleansing bar with N-cocoyl L-proline and N-capryloyl L-glutamine
To a stirred mixture of hydrogenated fatty acid triglycerides (360 g, 0.45 gmol), and glycerin (40 g, 0.43 gmol) at 90-95 ? under nitrogen blanket in a jacketed sigma blender (with ‘Z’ type blades that rotate in opposite directions to each other), sodium cocoyl isethionate (powder 85 % active, particle size < 250 µm, 450 g, 1.10 gmol) is added slowly (about two hours) and stirring is continued for three hours. The mass in the sigma blender is cooled to 65 ? under continuous kneading and to it, L-Glutamic acid, N, N-diacetic acid, tetrasodium salt, (2.0 g), Vitamin E acetate (5.0 g), polyglyceryl -3 oleate (90 g, 0.12 gmol), the blend of Example 8 (12 g, N-cocoyl L-proline and N-capryloyl L-glutamine), and water (50.0 g) are added and the kneading operation is continued at 60-65 ? for 4 hours. The mass is then cooled to 30 to 35 ? (993 g) while continuously kneading, refined using a triple roll mill and extruded using a duplex plodder (38-42 ?) to get the billets for stamping.
Table 6: Analysis of the bar cake:
Moisture 4. 0%
pH (5 % dispersion in water) 5.55
Active matter (as sodium cocoyl isethionate, MW 347) 39.07%
Hardness 8
Grit Absent
Mush 6.0 g /50 cm2

, Claims:
1. A cold-processable, bio-renewable, biodegradable and prebiotic preservative system for personal care compositions, comprising:
i. N-acyl L-proline of Formula I wherein R1CO is selected from fatty acyl chain of C8 to C18;
ii. N-acyl L-glutamine of Formula II wherein R2CO is selected from capryloyl or undecylenoyl;

Formula I

Formula II
wherein the molar ratio of N-acyl L-proline of Formula I, and N-acyl L-glutamine of Formula II, is from 2:1 to 1:2; and
iii. water, comprising of 20 to 30 % by weight of the total preservative composition.

2. The cold-processable, bio-renewable, biodegradable and prebiotic preservative system for personal care compositions as claimed in claim 1 wherein, the N-acyl L-amino acids are manufactured in one pot sequentially by reacting fatty acid chlorides with the corresponding amino acids in the presence of base.

3. A personal care composition comprising:
i. N-acyl L-proline of Formula I wherein R1CO is fatty acyl chain of C8 to C18;
ii. N-acyl L-glutamine of Formula II wherein R2CO is selected from capryloyl or undecylenoyl; and

Formula I

Formula II

iii. one or more personal care ingredient,
wherein, the personal care composition is devoid of petrochemical based antimicrobial preservatives.

4. The personal care composition as claimed in claim 3, wherein the N-acyl L-proline of Formula I and the N-acyl L-glutamine of Formula II is in the range of 0.5 to 4.0 % by weight of the personal care composition.

5. The personal care composition as claimed in claim 3, wherein the pH ranges from 3.0 to 6.0.

6. The personal care composition as claimed in claim 3 to 5 wherein the personal care composition is transparent or opaque.

7. The personal care composition as claimed in claim 3 to 6, wherein the personal care composition is selected from a shampoo, a body wash, a face wash, a hair conditioner, a hair serum, a soap bar, a syndet bar for hair and skin, a cream, a lotion, a hygiene wash, a deodorant, an anti-perspirant and a gel.

8. The personal care composition as claimed in claim 3 to 7, wherein the one or more personal care ingredient is selected from anionic surfactants, amphoteric surfactants, non-ionic surfactants, cationic surfactants, pearlisers, opacifiers, emollients, anti-inflammatory, bio-renewable anti-microbial actives, UV-absorbers, UV-blockers, synthetic and/or bio-based polymeric conditioners, silicones, gums, rheology modifiers, polymeric rheology modifiers, film formers, vitamins, protein derivatives, anti-acne agents, anti-dandruff agents, chelating agents, anti-oxidants, skin care active, hair active, water and mixture thereof.

9. A method of preventing microbial contamination of a personal care composition, said method comprises the addition of 0.5 to 4 % by weight, based on the total weight of the personal care composition, of at least one compound of formula I and at least one compound of Formula II,

Formula I

Formula II
wherein R1CO in Formula I is selected from fatty acyl chain of C8 to C18 and R2CO in formula II is selected from capryloyl or undecylenoyl.

10. A method of preventing microbial contamination as claimed in claim 9, wherein the personal care composition is selected from shampoo, body wash, face wash, hair conditioner, hair serum, soap bar, syndet bar for hair and skin, cream, lotion, hygiene wash, deodorant, anti-perspirant and gel.

11. A method of preventing microbial contamination as claimed in claim 9, wherein the personal care composition has pH of between 3 to 6.

12. A method of preventing microbial contamination as claimed in claim 9, wherein ratio of the compound of formula I and the compound of formula II is between 1:2 and 2:1.