COSMETIC COMPOSITION COMPRISING A POLYHYDROXYALKANOATE COPOLYMER BEARING A(N) (UN)SATURATED HYDROCARBON-BASED CHAIN AND A SURFACTANT
20230120675 · 2023-04-20
Inventors
Cpc classification
A61K8/4993
HUMAN NECESSITIES
A61K8/39
HUMAN NECESSITIES
A61Q1/00
HUMAN NECESSITIES
A61K8/894
HUMAN NECESSITIES
International classification
A61K8/39
HUMAN NECESSITIES
A61K8/894
HUMAN NECESSITIES
Abstract
The present invention relates to a cosmetic composition comprising a) one or more polyhydroxyalkanoate (PHA) copolymers which contain, and preferably consist of, at least two different repeating polymer units chosen from the units (A) and (B) below, and also the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and the solvates thereof such as hydrates: -[-0-CH(R.sup.1)—CH.sub.2—C(0)-]- unit (A) -[-0-CH(R.sup.2)—CH.sub.2—C(0)-]- unit (B) in which polymer units (A) and (B): —R.sup.1 represents a hydrocarbon-based chain chosen from i) linear or branched (C.sub.5-C.sub.28)alkyl, ii) linear or branched (C.sub.6-C.sub.28)alkenyl, iii) linear or branched (C.sub.6-C.sub.28)alkynyl; preferably, the hydrocarbon-based group is linear; said hydrocarbon-based chain being optionally substituted and/or interrupted with atoms or groups as described in the description; —R.sup.2 represents a cyclic or non-cyclic, linear or branched, saturated or unsaturated hydrocarbon-based group, comprising from 3 to 30 carbon atoms; and b) one or more surfactants; it being understood that (A) is different from (B).
Claims
1. A composition comprising: a) one or more polyhydroxyalkanoate (PHA) copolymers which contain, at least two different repeating polymer units chosen from the units (A) and (B) below, and the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and the solvates thereof:
—[—O—CH(R.sup.1)—CH.sub.2—C(O)—]— unit (A)
—[—O—CH(R.sup.2)—CH.sub.2—C(O)—]— unit (B) in which polymer units (A) and (B): R.sup.1 represents a hydrocarbon-based chain chosen from i) linear or branched (C.sub.5-C.sub.28)alkyl, ii) linear or branched (C.sub.5-C.sub.28)alkenyl, iii) linear or branched (C.sub.5-C.sub.28)alkynyl; said hydrocarbon-based chain being: optionally substituted with one or more atoms or groups chosen from: a) halogen, b) hydroxyl, c) thiol, d) (di)(C.sub.1-C.sub.4)(alkyl)amino, e) (thio)carboxy, f) (thio)carboxamide —C(O)—N(R.sub.a).sub.2 or C(S)—N(R.sub.a).sub.2, g) cyano, h) iso(thio)cyanate, i) (hetero)aryl and j) (hetero)cycloalkyl, k) cosmetic active agent; l) R—X with R representing a group chosen from α) cycloalkyl, β) heterocycloalkyl, γ) (hetero)aryl, δ) cosmetic active agent as defined previously and X representing a′) O, S, N(R.sub.a) or Si(R.sub.b)(R.sub.c), b′) S(O).sub.r, or (thio)carbonyl, c′) or combinations of a′) with b′); R.sub.a representing a hydrogen atom, or a (C.sub.1-C.sub.4)alkyl group or an aryl(C.sub.1-C.sub.4)alkyl group; R.sub.b and R.sub.c, which may be identical or different, represent a (C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)alkoxy group; and/or optionally interrupted with one or more heteroatoms a′) O, S, N(R.sub.a) or Si(R.sub.b)(R.sub.c), b′) S(O).sub.r, (thio)carbonyl, c′) or combinations of a′) with b′) with r being equal to 1 or 2, R.sub.a being as defined previously, R.sub.b and R.sub.c being as defined previously; R.sup.2 represents a cyclic or non-cyclic, linear or branched, saturated or unsaturated hydrocarbon-based group comprising from 3 to 30 carbon atoms; and b) one or more surfactant(s); and c) optionally one or more fatty substances; it being understood that (A) is different from (B).
2. The composition according to claim 1, in which the PHA copolymer(s) a) contain the repeating unit of formula (I), and the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and the solvates thereof: ##STR00052## in which formula (I): m and n are integers greater than or equal to 1.
3. The composition according to claim 1, in which the PHA copolymer(s) a) contain three different repeating polymer units (A), (B) and (C), and preferably consist of three different polymer units (A), (B) and (C), below, and the optical or geometrical isomers thereof and the solvates thereof:
—[—O—CH(R.sup.1)—CH.sub.2—C(O)—]— unit (A)
—[—O—CH(R.sup.2)—CH.sub.2—C(O)—]— unit (B)
—[—O—CH(R.sup.3)—CH.sub.2—C(O)—]— unit (C) in which polymer units (A), (B) and (C): R.sup.3 represents a cyclic or non-cyclic, linear or branched, saturated or unsaturated hydrocarbon-based group comprising from 1 to 30 carbon atoms; and it being understood that: (A) is different from (B) and (C), (B) is different from (A) and (C), and (C) is different from (A) and (B).
4. The composition according to claim 1, in which the PHA copolymer(s) a) contain four different repeating polymer units (A), (B), (C) and (D), below, and the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and also the solvates thereof:
—[—O—CH(R.sup.1)—CH.sub.2—C(O)—]— unit (A)
—[—O—CH(R.sup.2)—CH.sub.2—C(O)—]— unit (B)
—[—O—CH(R.sup.3)—CH.sub.2—C(O)—]— unit (C)
—[—O—CH(R.sup.4)—CH.sub.2—C(O)—]— unit (D) in which polymer units (A), (B), (C) and (D): R.sup.4 represents a cyclic or non-cyclic, linear or branched, saturated hydrocarbon-based group comprising from 3 to 30 carbon atoms optionally substituted with one or more atoms or groups a) to l) and/or optionally interrupted with one or more heteroatoms or groups a′) to c′) as defined for R.sup.1; and it being understood that: (A) is different from (B), (C) and (D), (B) is different from (A), (C) and (D), (C) is different from (A), (B) and (D), and (D) is different from (A), (B) and (C) in which formula (III).
5. The composition according to claim 1, in which the PHA copolymer(s) a) contain five different repeating polymer units (A), (B), (C), (D) and (E), and preferably consist of five different polymer units (A), (B), (C), (D) and (E), below, and the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and also the solvates thereof:
—[—O—CH(R.sup.1)—CH.sub.2—C(O)—]— unit (A)
—[—O—CH(R.sup.2)—CH.sub.2—C(O)—]— unit (B)
—[—O—CH(R.sup.3)—CH.sub.2—C(O)—]— unit (C)
—[—O—CH(R.sup.4)—CH.sub.2—C(O)—]— unit (D)
—[—O—CH(R.sup.5)—CH.sub.2—C(O)—]— unit (E) in which polymer units (A), (B), (C), (D) and (E): R.sup.5 represents a cyclic or non-cyclic, linear or branched, saturated hydrocarbon-based group comprising from 3 to 30 carbon atoms optionally substituted with one or more atoms or groups a) to l) and/or optionally interrupted with one or more heteroatoms or groups a′) to c′) as defined for R.sup.1; it being understood that: (A) is different from (B), (C), (D) and (E); (B) is different from (A), (C), (D) and (E); (C) is different from (A), (B), (D) and (E); (D) is different from (A), (B), (C) and (E); and (E) is different from (A), (B), (C) and (D).
6. The composition according to claim 1, in which R.sup.1 represents a linear or branched (C.sub.5-C.sub.28)alkyl hydrocarbon-based chain.
7. The composition according to claim 1, in which R.sup.1 represents a hydrocarbon-based chain.
8. The composition according to claim 1, in which R.sup.1 has the following formula —(CH.sub.2).sub.r—X-(ALK).sub.u-G with X being as defined previously, ALK represents a linear or branched (C.sub.1-C.sub.10)alkylene, r represents an integer inclusively between 6 and 11; and G represents a hydrogen atom or a group chosen from hydroxyl, carboxyl, (di)(C.sub.1-C.sub.4)(alkyl)amino, (hetero)aryl, cycloalkyl, or a sugar.
9. The composition according to claim 1, in which the PHA copolymer(s) a) are such that R.sup.2 is chosen from linear or branched (C.sub.1-C.sub.28)alkyl, and linear or branched (C.sub.2-C.sub.28)alkenyl.
10. The composition according to claim 1, in which the PHA copolymer(s) a) are such that the radical R.sup.2 is a linear (C.sub.1-C.sub.8)alkyl group; or R.sup.2 is a branched (C.sub.3-C.sub.8)alkyl group.
11. The composition according to claim 1, in which the PHA copolymer(s) a) are such that: the unit (A) is present in a molar percentage ranging from 0.1% to 99; and the unit (B) is present in a molar percentage ranging from 1% to 40; and/or the unit (C) is present in a molar percentage ranging from 0.5% to 20%.
12. (canceled)
13. The composition according to claim 1, in which b) the surfactant(s) are ionic; and/or the surfactant(s) b) have a HLB value greater than 10.
14. The composition according to claim 1, which is a direct emulsion.
15. The composition according to claim 1, which is an inverse emulsion.
16. The composition according to claim 1, in which the fatty medium comprises one or more substances chosen from: branched C.sub.8-C.sub.16 alkanes, linear C.sub.8-C.sub.16 alkanes; ester oils; monoester oils of formula R.sup.9—C(O)—OR.sup.10 in which R.sup.9 represents a linear or branched hydrocarbon-based chain including from 5 to 19 carbon atoms and R.sup.10 represents a linear or branched, notably branched, hydrocarbon-based chain containing from 4 to 20 carbon atoms, on condition that R.sup.9+R.sup.10≥9 carbon atoms; esters of lactic acid and of C.sub.10-C.sub.20 alcohol; diesters of malic acid and of C.sub.10-C.sub.20 alcohol; esters of pentaerythritol and of C.sub.8-C.sub.22 carboxylic acid; diesters of formula R.sup.11—O—C(═O)—R.sup.12—C(═O)—O—R.sup.13, with R.sup.11 and R.sup.13, which may be identical or different, representing a linear or branched, saturated or unsaturated C.sub.4 to C.sub.12 C.sub.5 to C.sub.10 alkyl chain, optionally containing at least one saturated or unsaturated ring, and R.sup.12 representing a saturated or unsaturated C.sub.1 to C.sub.4, alkylene chain; diesters of formula R.sup.14—C(═O)—O—R.sup.15—O—C(═O)—R.sup.16, with R.sup.14 and R.sup.16, which may be identical or different, representing a linear or branched, saturated or unsaturated C.sub.4 to C.sub.12 alkyl chain and R.sup.15 representing a saturated or unsaturated C.sub.1 to C.sub.4 alkylene chain; the carbonate oils being chosen from the carbonates of the following formula R.sup.17—O—C(O)—O—R.sup.18, with R.sup.17 and R.sup.18, which may be identical or different, representing a linear or branched C.sub.4 to C.sub.12 alkyl chain; and mixtures thereof.
17. The composition according to claim 1, in which the fatty medium comprises one or more fatty substances in a content ranging from 2% to 99.9% by weight, relative to the total weight of the composition.
18. The composition according to claim 1, in which the fatty medium comprises one or more solvents.
19. The composition according to claim 1, which further comprises one or more colouring agents chosen from pigments, direct dyes and mixtures thereof.
20. A process for treating keratin materials by applying the composition as defined in claim 1.
21. (canceled)
22. The composition according to claim 1, in which the PHA copolymer(s) a) are such that they comprise the following repeating units, and the optical or geometrical isomers thereof, the organic or mineral acid or base salts thereof, and the solvates thereof: TABLE-US-00028
Description
EXAMPLES
[0631] The PHAs illustrated in the various examples were prepared in 3-litre chemostats and/or 5-litre Fernbach flasks depending on whether or not a p-oxidation pathway inhibitor is used. The isolation of the PHAs is similar for all the examples obtained.
[0632] In a first step, the microorganism generates the PHAs which are stored in intracellular granules, the proportion of which varies as a function of the applied conditions such as the temperature or the nature of the culture medium. The generation of PHA granules may or may not be associated with the growth of the microorganism as a function of the nature of the microorganisms. During the second step, the biomass containing the PHAs is isolated, i.e. separated from the fermentation medium, and then dried. The PHAs are extracted from the biomass before being purified, if necessary.
[0633] A mixture of saturated and unsaturated carbon sources is, for certain examples, necessary for the stability of the PHA obtained.
TABLE-US-00008 TABLE 5 Carbon source CAB Caprylic acid RABIACID 308; 124-07-2 Nonanoic acid 112-05-0 Undecylenic acid 112-38-9 (10-Undecencicacid)
TABLE-US-00009 TABLE 6 Carbon source Genus and species Source Caprylic and undecylenoic Pseudomonas ATCC ® acid mixture putids 47054 ™ Nonanoic and undecylenoic Pseudomonas ATCC ® acid mixture putids 47054 ™
Example 1: PHA Bearing a Side Chain R.SUP.1 .Representing a Linear 10% Unsaturated n-Octenyl Group and R.SUP.2 .Representing an n-Pentyl Group
[0634] ##STR00032##
[0635] The process for synthesizing the compound of Example 1 is adapted from the article: Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by Pseudomonas putida KT2440, Z. Sun, J. A. Ramsay, M. Guay, B. A. Ramsay, Applied Microbiology Biotechnology, 82, 657-662, 2009.
[0636] The microorganism used is Pseudomonas putida KT2440 ATCC® 47054™. The culture method is performed under fed-batch growth axenic conditions with a maintenance solution containing a mixture of carbon source at a rate μ=0.15 h.sup.−1 in a 3 L chemostat containing 2.5 L of culture medium.
[0637] The system is aerated with a flow of 0.5 vvm of air for a nominal dissolved oxygen (O.sub.D) value at 30% of saturation. The pH is regulated with 15% aqueous ammonia solution. The temperature of the fermentation medium is regulated at 30° C.
[0638] Assembly for the Fed-Batch Growth Fermentation Mode
[0639] The fermentation medium is regulated in terms of temperature-pressure of dissolved oxygen and pH (not shown)
[0640] See
[0641] The production process is performed using three different culture media. The first culture medium, defined CM1 “inoculum”, is used for the preparation of the preculture. The second culture medium, defined CM2 “batch”, is used for unfed batch growth of the microorganism with the primary carbon sources in the Fernbach flasks. The third culture medium, defined CM3 “maintenance”, is used for the fed-batch or maintenance fermentation mode with the carbon sources of interest at a flow rate calibrated as a function of the growth of the microorganism.
TABLE-US-00010 TABLE 7 Ingredients in grams CN1 CM2 CM 3 per litre << incoulum >> << batch >> << maitenance >> (NH.sub.4).sub.2SO.sub.4 47 4.7 Ns.sub.2HPO.sub.4 .Math. 7H.sub.2O 12 9 KH.sub.2PO.sub.4 2.7 2.03 MgSO.sub.4 .Math. 7H.sub.2O 0.8 1.03 Nutrient Broth 3 / Caprylic acid / 0.9 900 Undecylenic acid / 0.1 100 Microelement solution / 10 Acrylic acid / / 2N NaOH QSP pH = 6.8 MilliQ water QSP 1000 g
[0642] The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™.
TABLE-US-00011 TABLE 8 Ingredients in grams per litre Amount FeSO.sub.4 .Math. 7H.sub.2O 10.0 g CaCl.sub.2 .Math. 2H.sub.2O 3.0 g ZnSO.sub.4 .Math. 7H.sub.2O 2.2 g MnSO.sub.4 .Math. 4H.sub.2O 0.5 g H.sub.3BO.sub.3 0.3 g CoCl.sub.2 .Math. 6H.sub.2O 0.2 g Na.sub.2MoO.sub.4 .Math. 2H.sub.2O 0.15 g NiCl.sub.2 .Math. 6H.sub.2O 0.02 g CuSO.sub.4 .Math. 5H.sub.2O 1.00 g MilliQ water QSP 1000 g (or 0.5N HCL)
[0643] 100 mL of preculture are prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of “inoculum” culture medium at a pH adjusted to 6.8 with 2N NaOH in a 250 mL Fernbach flask and are then incubated at 30° C. at 150 rpm for 24 hours. 1.9 L of CM2 “batch” culture medium placed in a presterilized 3 L chemostat are inoculated at OD=0.1 with the 100 mL of preculture, after 4 hours at 30° C. at 850 rpm.
[0644] At the end of the introduction, the biomass is isolated by centrifugation and then washed three times with water. The biomass is dried by lyophilization before being extracted with ethyl acetate for 24 hours. The suspension is clarified by filtration on a GF/A filter (Whatman®). The filtrate, the PHA compound dissolved in the ethyl acetate, is concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass.
[0645] The PHA may optionally be purified by successive dissolution and precipitation from an ethyl acetate/ethanol 70% methanol system, for example.
[0646] The PHA was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure.
Example 2: Poly(3-Hydroxyoctanoate-Co-Undecenoate) Containing 10% Unsaturations 100% Grafted with Thiolactic Acid (Compound of Example 1 Grafted with Thiolactic Acid TLA)
[0647] ##STR00033##
[0648] 1 g of the compound of Example 1 and 150 mg of thiolactic acid were dissolved in 20 mL of ethyl acetate at room temperature with stirring. 20 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0649] 20 mL of the reaction medium were then precipitated from a 200 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0650] The grafted PHA of Example 2 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure.
Example 3: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 100% Grafted with Octanethiol (Compound of Example 1 Grafted with n-octanethiol)
[0651] ##STR00034##
[0652] 0.5 g of the compound of Example 1 and 125 mg of octanethiol were dissolved in 10 mL of ethyl acetate at room temperature with stirring. 15 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0653] The reaction medium was then precipitated from a 100 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0654] The grafted PHA of Example 3 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure.
Example 4: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 75% Grafted with 8-mercapto-1-octanol (Compound of Example 1 Grafted with 8-mercapto-1-octanol)
[0655] ##STR00035##
[0656] 50 mg of the compound of Example 1 and 10 mg of 8-mercapto-1-octanol were dissolved in 5 mL of ethyl acetate at room temperature with stirring. 2 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0657] The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated.
[0658] The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0659] The grafted PHA of Example 4 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 75% or 7.5% of functions in total.
Example 5: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 32% Grafted with Cysteamine (Compound of Example 1 Grafted with Cysteamine)
[0660] ##STR00036##
[0661] 0.5 g of the compound of Example 1 and 54 mg of cysteamine were dissolved in a mixture of 10 mL of dichloromethane and 2 mL of ethanol at room temperature with stirring. 10 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0662] The reaction medium was then precipitated from a 100 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0663] The grafted PHA of Example 5 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 32% (see the spectrum below) or 3.2% of functions in total.
Example 6: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 73% Grafted with Cyclohexanethiol (Compound of Example 1 Grafted with CHT)
[0664] ##STR00037##
[0665] 100 mg of the compound of Example 1 and 26 mg of cyclohexanethiol were dissolved in 5 mL of dichloromethane at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0666] The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0667] The grafted PHA of Example 6 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 73% or 7.3% of functions in total.
Example 7: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 66% Grafted with 2-furanmethanethiol (FT) (Compound of Example 1 Grafted with FT)
[0668] ##STR00038##
[0669] 100 mg of the compound of Example 1 and 26 mg of 2-furanmethanethiol were dissolved in 5 mL of dichloromethane at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0670] The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0671] The grafted PHA of Example 7 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 66% or 6.6% of functions in total.
Example 8: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 66% Grafted with 1-thio-β-D-glucose tetraacetate (Compound of Example 1 Grafted with TGT)
[0672] ##STR00039##
[0673] 100 mg of the compound of Example 1 and 26 mg of 1-thio-β-D-glucose tetraacetate were dissolved in 5 mL of dichloromethane at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture.
[0674] The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes. The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated.
[0675] The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0676] The grafted PHA of Example 8 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 70% or 7% of functions in total.
Example 9: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 50% Grafted with 2-phenylethanethiol (PT) (compound of Example 1 grafted with PT)
[0677] ##STR00040##
[0678] 100 mg of the compound of Example 1 and 26 mg of 2-phenylethanethiol were dissolved in 5 mL of dichloromethane at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0679] The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0680] The grafted PHA of Example 9 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 50% or 5% of functions in total.
Example 10: Poly(3-hydroxyoctanoate-co-undecenoate) Containing 10% Unsaturations 64% Grafted with 4-tert-butylbenzyl mercaptan (TBM) (Compound of Example 1 Grafted with TBM)
[0681] ##STR00041##
[0682] 100 mg of the compound of Example 1 and 26 mg of 4-tert-butylbenzyl mercaptan were dissolved in 5 mL of dichloromethane at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture.
[0683] The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes. The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated.
[0684] The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0685] The grafted PHA of Example 10 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 64% or 6.4% of functions in total.
Example 11: Poly(3-hydroxynonanoate-co-undecenoate) Containing 10% Unsaturations 100% Grafted with Thiolactic Acid
[0686] ##STR00042##
[0687] 0.1 g of the compound of Example 1 and 15 mg of thiolactic acid were dissolved in 5 mL of chloroform at room temperature with stirring. 5 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0688] The reaction medium was then precipitated from a 50 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0689] The grafted PHA of Example 11 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 100%.
Example 12: Poly(3-hydroxynonanoate-co-undecenoate) Containing 5% Unsaturations 100% Grafted with Octanethiol
[0690] ##STR00043##
Preparation of Example 1: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an n-Hexyl Group and R.SUP.2 .Representing an n-Hexyl Group
[0691] ##STR00044##
[0692] The production process of Example 1 is adapted to that of Example 1′, replacing the n-octanoic acid carbon source of Example 1 with n-nonanoic acid.
[0693] The PHA copolymer of Example 1′ was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure, with a degree of unsaturation of 5%.
[0694] 1 g of the PHA copolymer of Example 1′ and 150 mg of octanethiol were dissolved in 15 mL of ethyl acetate at room temperature with stirring. 20 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0695] The reaction medium was then precipitated from a 500 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0696] The grafted PHA of Example 12 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 100%.
Example 13: Poly(3-hydroxynonanoate-co-undecenoate) Containing 5% Unsaturations 100% Epoxidized
[0697] ##STR00045##
[0698] 20 g of the PHA copolymer of Example 1′ were dissolved in 80 mL of anhydrous dichloromethane. A suspension of 1.9 g of 77% m-CPBA was prepared with 20 mL of anhydrous dichloromethane and added to the mixture with stirring, at room temperature for at least 120 hours.
[0699] The reaction medium was then precipitated from a 500 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0700] The PHA of Example 13 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Epoxidation to 100%.
Example 14: Poly(3-hydroxynonanoate-co-undecenoate) Containing 10% Unsaturations 100% Epoxidized
[0701] 10 g of the PHA copolymer identical to that of Example 1′ but with a degree of unsaturation of 10% were dissolved in 40 mL of anhydrous dichloromethane. A suspension of 1.9 g of 77% m-CPBA was prepared with 10 mL of anhydrous dichloromethane and added to the mixture with stirring, at room temperature for at least 120 hours.
[0702] The reaction medium was then precipitated from a 500 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0703] The PHA of Example 14 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Epoxidation to 100%.
Example 15: Poly(3-hydroxynonanoate-co-undecenoate) Containing 30% Unsaturations 100% Epoxidized
[0704] 10 g of the PHA copolymer identical to that of Example 1′ but with a degree of unsaturation of 30% were dissolved in 40 mL of anhydrous dichloromethane. A suspension of 6.2 g of 77% m-CPBA was prepared with 10 mL of anhydrous dichloromethane and added to the mixture with stirring, at room temperature for at least 120 hours.
[0705] The reaction medium was then precipitated from a 250 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0706] The PHA of Example 15 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Epoxidation to 100%.
Example 16: Poly(3-hydroxynonanoate-co-undecenoate) Containing 5% Unsaturations 100% Grafted with 4-tert-butylbenzyl mercaptan (TBM) (Compound of Example 1′ Grafted with TBM)
[0707] ##STR00046##
[0708] 2 g of the PHA copolymer of Example 1′ and 300 mg of 4-tert-butylbenzyl mercaptan were dissolved in 25 mL of ethyl acetate at room temperature with stirring. 25 mg of 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651) were added to the mixture. The medium was then irradiated under a 100 W UV lamp at 365 nm (reference) and with stirring for at least 10 minutes.
[0709] The reaction medium was then precipitated from a 500 mL mixture of 70/30 v/v ethanol/water. A viscous white precipitate was obtained. This step may be repeated. The product thus obtained was dissolved in a minimum amount of ethyl acetate, poured onto a Teflon plate and then dried under dynamic vacuum at 40° C. to obtain a homogeneous film.
[0710] The PHA of Example 16 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure. Grafting to 100%.
Example 17: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an Isohexenyl Group and R.SUP.2 .Representing an Isobutyl Group
[0711] ##STR00047##
[0712] The production process of Example 3 is an adaptation of Applied and Environmental Microbiology, Vol. 60, No. 9. 3245-3254 (1994) “Polyester Biosynthesis Characteristics of Pseudomonas citronellolis Grown on Various Carbon Sources, Including 3-Methyl-Branched Substrate”. Mun Hwan Choi and Sung Chul Yoon. The microorganism used is Pseudomonas citronellolis ATCC® 13674™. The culture method is performed under unfed-batch axenic culture conditions in 5 L Fernbach flasks (Corning® ref. 431685) containing 2 L of culture medium, shaken at 110 rpm at 30° C. in an orbital incubator (diameter of the orbit of 2.5 cm).
[0713] The production process is performed using two different culture media. The first culture medium, defined CM1 “inoculum”, is used for the preparation of the preculture. The second culture medium, defined CM2 “batch”, is used for unfed batch culture growth of the microorganism with the carbon source of interest in the Fernbach flasks.
TABLE-US-00012 TABLE 9 Ingredients in grams per litre CM1 « inoculum » CM2 « batch » (NH4).sub.2SO.sub.4 / 0.66 Na.sub.2HPO.sub.4•7H.sub.2O / 7.3 KH.sub.2PO.sub.4 / 2.3 NaHCO.sub.3 / 0.3 CaCl.sub.2•2H.sub.2O / 0.1 MgSO.sub.4•7H.sub.2O / 0.25 Citric acid / 1.03 Citronellol / 5.5 Microelement solution / 1 Nutrient broth 1.5 / Yeast extract 1 / 2N NaOH QSP pH = 6.8 MilliQ water QSP m = 1000 g
[0714] The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™ ED.
[0715] The composition of the yeast extract, as a mass percentage, is 100% autolysate of the yeast Saccharomyces cerevisiae. Reference 210933 DIFCO™ ED.
TABLE-US-00013 TABLE 10 Ingredients in grams per litre Amount FeSO.sub.4•7H.sub.2O 5.56 g CaCl.sub.2•2H.sub.2O 3.0 g ZnSO.sub.4•7H.sub.2O 0.58 g MnCl.sub.2•4H.sub.2O 3.86 g H.sub.3BO.sub.3 0.6 g CoCl.sub.2•6H.sub.2O 5.62 g Na.sub.2MoO.sub.4•2H.sub.2O 0.06 g NiCl.sub.2•6H.sub.2O 0.04 g CuSO.sub.4•5H.sub.2O 0.34 g HCl 0.5N QSP 1000 g
[0716] 100 mL of preculture are prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of “inoculum” culture medium at a pH adjusted to 6.8 with 2N NaOH in a 250 mL Fernbach flask and then incubated at 30° C. at 150 rpm for 24 hours. 1.9 L of CM2 “batch” culture medium placed in a presterilized 5 L Fernbach flask are inoculated at OD=0.1 with 100 mL of inoculum.
[0717] After 70 hours at 30° C. at 110 rpm, the biomass is dried by lyophilization before being extracted with dichloromethane for 24 hours. The suspension is clarified by filtration on a GF/A filter (Whatman®). The filtrate, composed of PHA dissolved in dichloromethane, is concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass.
[0718] The PHA may optionally be purified by successive dissolution and precipitation, for instance using a dichloromethane/methanol system.
[0719] The PHA copolymer of Example 3 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure, with: 68 mol % of unit (A) for which R.sup.1=isohexenyl and 32 mol % of unit (B) for which R.sup.2=isobutyl.
Example 18: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an Isohexyl Group and R.SUP.2 .Representing an Isobutyl Group
[0720] ##STR00048##
[0721] Example 18 is obtained by hydrogenation of Example 17 using an H-Cube Midi® continuous hydrogenator from ThalesNano Technologies.
[0722] A solution of 2 g (8.83 mmol) of Example 3 is prepared with a mixture composed of 100 mL of ethyl acetate (Sigma-Aldrich-CAS: 141-78-6) and 100 mL of methanol (Sigma-Aldrich-CAS: 67-56-1) is introduced at a flow rate of 3 mL per minute into a hydrogenation cartridge containing the catalyst containing 5% palladium on charcoal (MidiCard ref. DHS 2141; ThalesNano Technologies) maintained at 100° C. under a pressure of 80 bar in the presence of hydrogen in the ThalesNano Technologies H-Cube Midi® system. The reduction of the double bond is monitored by NMR. After six consecutive cycles of reduction, the solution is concentrated by evaporation and then dried under vacuum to constant mass.
[0723] The PHA may optionally be purified by successive dissolution and precipitation, for instance using a dichloromethane/methanol system.
[0724] The PHA copolymer of Example 4 was fully characterized by spectroscopic and spectrometric methods and is in accordance with the expected chemical structure, with: 68 mol % of unit (A) for which R.sup.1=isohexyl and 32 mol % of unit (B) for which R.sup.2=isobutyl.
Example 19
[0725] A polymer was prepared using the microorganism Pseudomonas putida KT2440 ATCC® 47054™, octanoic acid.
[0726] The culture method was performed under batch axenic conditions in 5 L Fernbach flasks (Corning® ref. 431685) containing 2 L of culture medium, shaken at 110 rpm at 30° C. in an orbital incubator (diameter of the orbit of 2.5 cm).
[0727] The synthetic process was performed using two different culture media. The first culture medium, defined CM1 “inoculum”, was used for the preparation of the inoculum. The second culture medium, defined CM2 “batch”, was used for unfed batch growth of the microorganism with the octanoic acid in the Fernbach flasks.
[0728] The composition in grams per litre of the two media is described in Table 11 below:
TABLE-US-00014 TABLE 11 CM1 CM2 « inoculum » « batch » (NH.sub.4).sub.2SO.sub.4 4.7 5.02 Na.sub.2HPO.sub.4•7H.sub.2O 12 2.24 KH.sub.2PO.sub.4 2.7 0.5 Glucose 9 3.9 MgSO.sub.4•7H.sub.2O 0.8 1.03 Citric acid 1.6 1.03 Nutrient Broth (1) 1 / Octanoic acid / 3.8 Microelement solution (2) / 1.4 2N NaOH QSP pH = 6.8 Water QSP 1000 g
[0729] (1) The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™.
[0730] (2) The composition of the microelement solution in grams per litre is described in Table 12 below:
TABLE-US-00015 TABLE 12 FeSO.sub.4•7H.sub.2O 10.0 g CaCl.sub.2•2H.sub.2O 3.0 g ZnSO.sub.4•7H.sub.2O 2.2 g MnSO.sub.4•4H.sub.2O 0.5 g H.sub.3BO.sub.3 0.3 g CoCl.sub.2•6H.sub.2O 0.2 g Na.sub.2MoO.sub.4•2H.sub.2O 0.15 g NiCl.sub.2•6H.sub.2O 0.02 g CuSO.sub.4•5H.sub.2O 1.00 g 0.5N HCL QSP 1000 g
[0731] 100 mL of inoculum were prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of “inoculum” culture medium at a pH adjusted to 6.8 with 2N NaOH in a 250 mL Fernbach flask and then incubated at 30° C. at 150 rpm for 24 hours. 1.9 L of CM2 “batch” culture medium placed in a presterilized 5 L Fernbach flask were inoculated at 00=0.1 with 100 mL of inoculum. After 70 hours at 30° C. at 110 rpm, the biomass was dried by lyophilization before being extracted with dichloromethane for 24 hours. The suspension was clarified by filtration on a GF/A filter (Whatman®). The filtrate, containing the copolymer dissolved in the dichloromethane, was concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass. The crude polyhydroxyalkanoate was purified by precipitation from a solution of the latter dissolved in 10 times its weight of dichloromethane, in 10 volumes of cold methanol solution. The solid obtained was dried under high vacuum at 40° C. to constant mass.
[0732] The molecular weight of the polyhydroxyalkanoate obtained was characterized by size exclusion chromatography, with detection by refractive index. [0733] Eluent: THF [0734] Analytical flow rate: 1 mL/min [0735] Injection: 100 μL [0736] Columns: 1 Agilent PLGel Mixed-D 5 μm column; 300×7.5 mm; 1 Agilent PLGel Mixed-C 5 μm column; 300×7.5 mm; 1 Agilent Oligopore column; 300×7.5 mm [0737] at room temperature (25° C.) [0738] Detection: Waters 2487 Dual I Absorbance Detector, Waters 2414 Refractive Index Detector [0739] Integrator: refractive index at 45° C. and 64 mV [0740] Empower (GC Relative molar mass/conventional module) [0741] Empower injection time: 40 min [0742] Standards: High mass/EasiVial PS-H 4 mL polystyrene from Agilent Technologies, Part No. PL2010-0200
[0743] The analysis makes it possible to measure the weight-average molecular weight (Mw in g/mol), the number-average molecular weight (Mn in g/mol), the polydispersity index Ip (Mw/Mn) and the degree of polymerization DPn.
[0744] The monomer composition of the polyhydroxyalkanoate obtained was defined by gas chromatography equipped with a flame ionization detector.
[0745] The identification is performed by injection of commercial standards and the monomer composition was determined by a methanolysis and silylation treatment.
[0746] To determine the monomer composition, 7 mg of the polyhydroxyalkanoate polymer were dissolved in 1.5 mL of chloroform and subjected to methanolysis in the presence of 1.5 mL of an MeOH/HCl solution (17/2, v/v) at 100° C. for 4 hours. The organic phase was then washed with 1 mL of water and then dried over MgSO4. Silylation of the methyl esters formed was performed by adding 100 μL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and 100 μL of pyridine to the methylated sample. The solution was heated at 70° C. for 1 hour and then evaporated to dryness. The sample was then dissolved in 600 μL of dichloromethane and analysed by chromatography under the following conditions: [0747] Hewlett Packard 6890 Series machine [0748] ZB-5 HT stationary phase column from Phenomenex (length: 30 m, diameter: 0.25 mm) [0749] Temperature: isotherm 60° C. to 300° C. in 6 min (heating rate: 10° C./min) [0750] Gas: Helium; flow rate: 0.8 mL/min [0751] Injector: Temperature: 250° C.; 50 ml/min [0752] Flame ionization detector; Temperature: 300° C. [0753] Injection: Volume 1 μL
[0754] A copolymer containing 91% by weight of poly(3-hydroxyoctanoate), 6% by weight of poly(3-hydroxyhexanoate) and 3% by weight of poly(3-hydroxybutanoate) was thus obtained. [0755] Mn=68 100 g/mol [0756] Mw=149 100 g/mol [0757] Ip=2.2 [0758] DPn=531
Example 2
[0759] A polymer was prepared using the microorganism Pseudomonas putida KT2440 ATCC® 47054™, octanoic acid and acrylic acid.
[0760] The culture method was performed under continuous axenic conditions at a dilution D=0.25 h.sup.−1 in a 3 L chemostat containing 1.1 L of culture medium. The system was aerated with air at a flow of 3 vvm (vvm=volume of air per volume of fermentation medium per minute) for a nominal dissolved oxygen (O.sub.D) value at 30% of saturation.
[0761] The production process was performed using three different culture media. The first defined culture medium (CM1) was used for the preparation of the inoculum. The second defined culture medium (CM2) was used for unfed batch growth of the microorganism in the fermenter. The third defined culture medium (CM3) was used for the feeding, or maintenance, of the continuous fermentation containing octanoic acid and acrylic acid (β-oxidation pathway inhibitor).
[0762] The media CM1 and CM2 are identical to those described in Example 1. The composition in grams per litre of the medium CM3 is described in Table 13 below:
TABLE-US-00016 TABLE 13 CM3 « continuous » (NH.sub.4).sub.2SO.sub.4 5.02 Na.sub.2HPO.sub.4•7H.sub.2O 2.24 KH.sub.2PO.sub.4 0.5 Glucose 3 MgSO.sub.4•7H.sub.2O 1.03 Citric acid 1.03 Nutrient Broth (1) / Octanoic acid 3.8 Microelement solution (2) 1.4 Acrylic acid 0.2 2N NaOH QSP pH = 6.8 Water Qsp 1000 g
[0763] 100 mL of inoculum were prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of Nutrient Broth at a pH adjusted to 7.0 with 2N NaOH in a 250 mL Fernbach flask and were then incubated at 30° C. at 150 rpm for 24 hours.
[0764] The fermenter containing 1 litre of culture medium CM2 at 30° C. was inoculated at an optical density of 0.1 at 630 nm (OD630=0.1). The system was maintained at 30° C. with shaking at 700±200 rpm and regulated in cascade with oxygenation for about 16 hours and/or the time for the microorganism to be able to reach its growth plateau.
[0765] Feeding of the fermenter with the medium CM3 was initiated when the microorganism reached its growth plateau, and withdrawal was then performed so as to maintain the initial mass of fermentation medium. Once the equilibrium state was reached in continuous culturing, a fraction of the withdrawn material was centrifuged so as to separate the biomass from the fermentation medium. The biomass was dried by lyophilization and then extracted with dichloromethane for 24 hours. The suspension obtained was clarified by filtration through a GF/A filter (Whatman®). The filtrate obtained, comprising the copolymer dissolved in dichloromethane, was concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass. The crude polyhydroxyalkanoate was purified by precipitation from a solution of the latter dissolved in 10 times its weight of dichloromethane, in 10 volumes of cold methanol solution. The solid obtained was dried under high vacuum at 40° C. to constant mass.
[0766] A copolymer comprising 96% by weight of poly(3-hydroxyoctanoate), 3% by weight of poly(3-hydroxyhexanoate) and 1% by weight of poly(3-hydroxybutanoate) was thus obtained. [0767] Mn=67 900 g/mol: [0768] Mw=142 000 g/mol: [0769] Ip=2.1: [0770] DPn=611
Example 20
[0771] A polymer was prepared according to the procedure of Example 2, using nonanoic acid (instead of octanoic acid).
[0772] A copolymer comprising 86% by weight of poly(3-hydroxynonanoate), 9% by weight of poly(3-hydroxyheptanoate) and 5% by weight of poly(3-hydroxypentanoate) was thus obtained. [0773] Mn=65 900 g/mol [0774] Mw=143 600 g/mol [0775] Ip=2.2 [0776] DPn=531
Example 21
[0777] A polymer was prepared according to the procedure of Example 2, using nonanoic acid (instead of octanoic acid) and without acrylic acid.
[0778] A copolymer comprising 68% by weight of poly(3-hydroxynonanoate), 27% by weight of poly(3-hydroxyheptanoate) and 5% by weight of poly(3-hydroxypentanoate) was thus obtained. [0779] Mn=55 800 g/mol [0780] Mw=124 500 g/mol [0781] Ip=2.2 [0782] DPn=469
Example 22
[0783] A polymer was prepared according to the procedure of Example 2, using dodecanoic acid (instead of octanoic acid).
[0784] A copolymer comprising 44% by weight of poly(3-hydroxydodecanoate), 38% by weight of poly(3-hydroxydecanoate) and 18% by weight of poly(3-hydroxyoctanoate) was thus obtained. [0785] Mn=67 400 g/mol [0786] Mw=129 800 g/mol [0787] Ip=1.9 [0788] DPn=484
Example 23: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an n-Pentyl Group and R.SUP.2 .Representing an n-Propyl Group
[0789] ##STR00049##
[0790] The production process of Example 23 is an adaptation of the article Biomacromolecules 2012, 13, 2926-2932: “Biosynthesis and Properties of Medium-Chain-Length Polyhydroxyalkanoates with Enriched Content of the Dominant Monomer”
[0791] The microorganism used is Pseudomonas putida ATCC® 47054™.
[0792] The culture method is performed under continuous axenic conditions at a dilution D=0.25 h.sup.−1 in a 3 L chemostat containing 1.1 L of culture medium.
[0793] The system is aerated with a flow of 3 vvm of air for a nominal dissolved oxygen (O.sub.D) value at 30% of saturation.
[0794] Assembly:
[0795] See
[0796] The production process is performed using three different culture media.
[0797] The first defined culture medium (CM1) is used for the preparation of the inoculum.
[0798] The second defined culture medium (CM2) is used for batch growth of the microorganism in the fermenter.
[0799] The third defined culture medium (CM3) is used for the feeding, or maintenance, of the continuous fermentation containing the carbon source of interest and the p-oxidation pathway inhibitor.
[0800] The composition in grams per litre of the three media is described in Table 14: composition in grams per litre of the culture media for the inoculum and the maintenance.
TABLE-US-00017 TABLE 14 CM1 CM2 CM3 « inoculum » « batch » « continuous » (NH4).sub.2SO4 4.7 5.02 5.02 Na.sub.2HPO4•7H2O 12 2.24 2.24 KH.sub.2PO4 2.7 0.5 0.5 Glucose 9 3.9 3 MgSO4•7H2O 0.8 1.03 1.03 Citric acid 1.6 1.03 1.03 Nutrient Broth 1 / / Octanoic acid / / 3.8 Microelement solution / 1.4 1.4 Acrylic acid / / 0.2 2N NaOH QSP pH = 0.8 Milli water QSP m = 1000 g
[0801] The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™.
[0802] The composition of the microelement solution in grams per litre is described in Table 15: composition in grams per litre of the microelement solution
TABLE-US-00018 TABLE 15 FeSO4•7H2O 10.0 g CaCl2•2H2O 3.0 g ZnSO4•7H2O 2.2 g MnSO4•4H2O 0.5 g H3BO3 0.3 g CaCl2•6H2O 0.2 g Na2MoO4•2H2O 0.15 g NiCl2•6H2O 0.02 g CuSO4•5H2O 1.00 g MilliQ QSP 1000 g
[0803] 100 mL of inoculum are prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of Nutrient Broth at a pH adjusted to 7.0 with 2N NaOH in a 250 mL Fernbach flask and are then incubated at 30° C. at 150 rpm for 24 hours.
[0804] The fermenter containing 1 litre of culture medium CM2 at 30° C. is inoculated at an optical density of 0.1 at 630 nm (OD630=0.1). The system is maintained at 30° C. with shaking at 700±200 rpm and regulated in cascade with oxygenation for about 16 hours and/or the time for the microorganism to be able to reach its growth plateau.
[0805] Feeding of the fermenter with the medium CM3 is initiated when the microorganism has reached its growth plateau, and withdrawal is then performed so as to maintain the initial mass of fermentation medium. Once the equilibrium state is reached in continuous culturing, a fraction of the withdrawn material is centrifuged so as to separate the biomass from the fermentation medium. The biomass is dried by lyophilization and is then extracted with dichloromethane for 24 hours. The suspension is clarified by filtration on a GF/A filter (Whatman®). The filtrate, composed of PHA dissolved in dichloromethane, is concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass.
[0806] The PHA may optionally be purified by successive dissolution and precipitation, for instance using a dichloromethane/methanol system.
[0807] The PHA copolymer of Example 23 was fully characterized by spectrometric and spectroscopic methods. By gas chromatography equipped with an FID detector, it is seen that the copolymer contains 96% of radical R.sup.1=n-pentyl and 4% of radical R.sup.2=n-propyl.
Example 24: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an n-Hexyl Group and R.SUP.2 .Representing an n-Butyl Group
[0808] ##STR00050##
[0809] The production process of Example 24 is an adaptation of the article Biomacromolecules, 13, 2926-2932 (2012): “Biosynthesis and Properties of Medium-Chain-Length Polyhydroxyalkanoates with Enriched Content of the Dominant Monomer”
[0810] The microorganism used is Pseudomonas putida ATCC® 47054™.
[0811] The culture method is performed under continuous axenic conditions at a dilution D=0.25 h.sup.−1 in a 3 L chemostat containing 1.1 L of culture medium.
[0812] The system is aerated with a flow of 3 vvm of air for a nominal dissolved oxygen (O.sub.D) value at 30% of saturation. The assembly is identical to that of the preceding example.
[0813] The composition in grams per litre of the three media is described in Table 16: composition in grams per litre of the culture media for the inoculum and the maintenance.
TABLE-US-00019 TABLE 16 CM1 CM3 « inoculum » CM2 « batch » « continuous » (NH4).sub.2SO4 4.7 5.02 5.02 Na.sub.2HPO4•7H2O 12 2.24 2.24 KH.sub.2PO4 2.7 0.5 0.5 Glucose 9 3.0 3.9 MgSO4•7H2O 0.8 1.03 1.03 Citric acid 1.6 1.03 1.03 Nurient Erod 1 / / Nonanoic acid / / 3.8 Microelement solution / 1.4 1.4 Acrylic acid / / 0.2 2N NaOH QSP pH = 6.8 MilliQ QSP m = 1000 g
[0814] The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™.
[0815] The composition of the microelement solution in grams per litre is described in Table 15.
[0816] 100 mL of inoculum are prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of Nutrient Broth at a pH adjusted to 7.0 with 2N NaOH in a 250 mL Fernbach flask and are then incubated at 30° C. at 150 rpm for 24 hours.
[0817] The fermenter containing 1 litre of culture medium CM2 at 30° C. is inoculated at an optical density of 0.1 at 630 nm (00630=0.1). The system is maintained at 30° C. with shaking at 700±200 rpm and regulated in cascade with oxygenation for about 16 hours and/or the time for the microorganism to be able to reach its growth plateau.
[0818] Feeding of the fermenter with the medium CM3 is initiated when the microorganism has reached its growth plateau, and withdrawal is then performed so as to maintain the initial mass of fermentation medium. Once the equilibrium state is reached in continuous culturing, a fraction of the withdrawn material is centrifuged so as to separate the biomass from the fermentation medium. The biomass is dried by lyophilization and is then extracted with dichloromethane for 24 hours. The suspension is clarified by filtration on a GF/A filter (Whatman®). The filtrate, composed of PHA dissolved in dichloromethane, is concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass.
[0819] The PHA copolymer of Example 24 may optionally be purified by successive dissolution and precipitation, for instance using a dichloromethane/methanol system.
[0820] The PHA copolymer of Example 24 was fully characterized by spectrometric and spectroscopic methods. By gas chromatography equipped with an FID detector, it is seen that the copolymer contains 86% of radical R.sup.1=n-hexyl and 14% of radical R.sup.2=n-butyl.
Example 25: Copolymer of PHA Bearing a Side Chain R.SUP.1 .Representing an n-Nonyl Group and R.SUP.2 .Representing an n-Heptyl Group
[0821] ##STR00051##
[0822] The production process of Example 25 is an adaptation of patent CA2781699C, Example 10.
[0823] The microorganism used is Pseudomonas putida ATCC® 47054™.
[0824] The culture method is performed under continuous axenic conditions at a dilution D=0.25 h.sup.−1 in a 3 L chemostat containing 1.1 L of culture medium.
[0825] The system is aerated with a flow of 3 vvm of air for a nominal dissolved oxygen (O.sub.D) value at 30% of saturation. The assembly is identical to that of Example 23.
[0826] The production process is performed using three different culture media.
[0827] The first defined culture medium (CM1) is used for the preparation of the inoculum.
[0828] The second defined culture medium (CM2) is used for batch growth of the microorganism in the fermenter.
[0829] The third defined culture medium (CM3) is used for the feeding, or maintenance, of the continuous fermentation containing the carbon source of interest and the β-oxidation pathway inhibitor.
[0830] The composition in grams per litre of the three media is described in Table 17: composition in grams per litre of the culture media for the inoculum and the maintenance.
TABLE-US-00020 TABLE 17 CM1 CM3 « inoculum » CM2 « batch » « continuous » (NH4).sub.2SO4 4.7 5.02 5.02 Na.sub.2HPO4•7H2O 12 2.24 2.24 KH.sub.2PO4 2.7 0.5 0.5 Glucose 9 3.0 3 MgSO4•7H2O 0.8 1.03 1.03 Citric acid 1.6 1.03 1.03 Nutrient Broth 1 / / Dodecanoic acid / / 1.14 Microelement solution / 1.4 1.4 Acrylic acid / / 0.2 2N NaOH QSP pH = 6.8 MilliQ QSP m = 1000 g
[0831] The composition of the Nutrient Broth, as mass percentages, is 37.5% beef extract and 62.5% peptone. Reference 233000 DIFCO™.
[0832] The composition of the microelement solution in grams per litre is described in Table 15.
[0833] 100 mL of inoculum are prepared by suspending a cryotube containing 1 mL of the strain with 100 mL of Nutrient Broth at a pH adjusted to 7.0 with 2N NaOH in a 250 mL Fernbach flask and are then incubated at 30° C. at 150 rpm for 24 hours. The fermenter containing 1 litre of culture medium CM2 at 30° C. is inoculated at an optical density of 0.1 at 630 nm (OD630=0.1). The system is maintained at 30° C. with shaking at 700±200 rpm and regulated in cascade with oxygenation for about 16 hours and/or the time for the microorganism to be able to reach its growth plateau. Feeding of the fermenter with the medium CM3 is initiated when the microorganism has reached its growth plateau, and withdrawal is then performed so as to maintain the initial mass of fermentation medium. Once the equilibrium state is reached in continuous culturing, a fraction of the withdrawn material is centrifuged so as to separate the biomass from the fermentation medium. The biomass is dried by lyophilization and is then extracted with dichloromethane for 24 hours. The suspension is clarified by filtration on a GF/A filter (Whatman®). The filtrate, composed of PHA dissolved in dichloromethane, is concentrated by evaporation and then dried under high vacuum at 40° C. to constant mass. The PHA copolymer may optionally be purified by successive dissolution and precipitation, for instance using a dichloromethane/methanol system.
[0834] The PHA copolymer of Example 25 was fully characterized by spectrometric and spectroscopic methods. By GC chromatography, it is seen that the copolymer contains 55% of radical R.sup.1=n-nonyl, 33% of radical R.sup.2=n-heptyl; 11% of radical R.sup.3=n-pentyl, 1.1% of R4=propyl and % R.sup.5=methyl.
[0835] Emulsification
Example 26: Direct Emulsion Using the PHA Copolymer of Example 23
[0836] Sodium laureth sulfate; sodium lauryl ether sulfate SLES (2.0 or 2.2 OE) anionic surfactants as an aqueous solution at 70% AM in water
[0837] 5 g of PHA copolymers of Example 23 are dissolved at room temperature with 45 g of ethyl acetate (EtOAc) before being dispersed with a solution of 50 g of SLES at 1% AM (0.7 g qs 50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm (revolutions per minute) in a jacketed reactor at 5° C. for 10 minutes. A temperature rise from 18° C. to 33° C. is observed.
[0838] The EtOAc is distilled off in successive stages on a bath at 45° C.
[0839] The mixture is made up to 35 g with water to obtain a white dispersion with blueish tints.
[0840] The dispersion is filtered at 0.45 μm under PSM.
Example 27: Direct Emulsion Using the PHA Copolymer of Example 24
[0841] The emulsion was prepared in the same manner as in the preceding example, starting with 5 g of copolymer of Example 24 instead of Example 23.
Example 28: Direct Emulsion Using the PHA Copolymer of Example 25
[0842] The emulsion was prepared in the same manner as in the preceding example, starting with 5 g of copolymer of Example 25 instead of Example 23.
Example 29: Inverse Emulsion Starting with the Copolymer of Example 24 (13% in the Fatty
[0843] Phase) Composition:
TABLE-US-00021 TABLE 18 Ingredients Amount in g/100g Copolymer of Example 24 4.80 CETYL PEG/PPC-10/1 DIMETHICONE (surfactant) 3.6 Poluglyceryl-4 isostearate (surfactant) 1.2 Isododecane 31.7 Butylene glycol 7.2 Magnesium sulfate 0.8 Denatured alcohol 9.5 Water Qsp 100
[0844] Protocol:
[0845] An aqueous phase is prepared by successive introduction of the ingredients of Table 8 with stirring at room temperature.
TABLE-US-00022 TABLE 19 Ingredients Amount in g Aqueous phase. Butylene glycol 7.2 Magnesium sulfate 0.8 Denatured alcohol 9.5 Water 41.2
[0846] The fatty phase is prepared in a 20 mL vial by introduction of the ingredients of Table 9 with stirring at 70° C. for 1 hour. The clear solution is brought to room temperature with stirring.
TABLE-US-00023 TABLE 20 Name of the ingredients Amount in g Fatty phase Surfactant CETYL PEG/PPG-10/1 3.6 (organic) DIMETHICONE POLYGLYCERYL-4 1.2 ISOSTEARATE Solvents ISODODECANE 31.7 PHA Example 24 4.8
[0847] 4.13 g of the fatty phase are diluted with 5.87 g of aqueous phase and the emulsion is prepared using an Ultra-Turrax blender (10 mm spindle) at 24 000 rpm for 10 minutes on a bath of ice-water.
[0848] The bright white and relatively fluid dispersion does not change in appearance after two weeks at room temperature (25° C.).
Example 30: Inverse Emulsion Starting with the Copolymer of Example 24 (28% in the Fatty Phase)
[0849] Composition:
TABLE-US-00024 TABLE 21 Ingredients Amount in g/100 g Copolymer of Example 24 10 CETYL PEG/PPG-10/1 DIMETHICONE 3.6 (surfactant) Polyglyceryl-4 isostearate (surfactant) 1.2 Isododecane 26.5 Butylene glycol 7.2 Magnesium sulfate 0.8 Denatured alcohol 9.5 Water Qsp 100
[0850] Protocol: Is Identical to that of the Preceding Example with the Same Aqueous Phase but a Different Amount of Fatty Phase:
TABLE-US-00025 TABLE 21 Name of the ingredients Amount in g Fatty phase Surfactant CETYL PEG/PPG-10/1 3.6 DIMETHICONE POLYGLYCERYL-4 1.2 ISOSTEARATE Solvents ISODODECANE 26.5 PHA Example 24 10
[0851] 4.13 g of the fatty phase are diluted with 5.87 g of aqueous phase and the emulsion is prepared using an Ultra-Turrax blender (10 mm spindle) at 24 000 rpm for 10 minutes on a bath of ice-water.
[0852] The bright white and relatively fluid dispersion does not change in appearance after two weeks at room temperature (25° C.).
EVALUATIONS
[0853] In a first stage, a film is prepared on a contrast card with a film spreader (speed: 50 mm/s-Cylinder: 100 μm). The film is left to dry for 24 hours at room temperature. Once dry, the film has a thickness of about 40 μm,
[0854] For the PHA copolymers of Examples 1 to 4 that are soluble in isododecane or an isododecane/ethanol mixture, evaluation of the cosmetic properties on a dry film was performed.
[0855] In a first stage, a film is prepared on a contrast card with a film spreader (speed: 50 mm/s-Cylinder: 100 μm). The film is left to dry for 24 hours at room temperature. Once dry, the film has a thickness of about 40 μm.
[0856] Three evaluations are performed on the dry film: Resistance to fats, gloss and tackiness
[0857] Measurement of the Resistance to Fats
[0858] Three drops of olive oil or sebum or water were deposited on the dry film present on the black part of the contrast card. Each drop corresponds to about 10 μL of olive oil (use of a micropipette).
[0859] The drop is left in contact with the dry film for two times: 5 minutes and 30 minutes. Once the time has elapsed, the drop of olive oil or sebum or water is wiped off and observation of the deterioration of the polymer film is performed. If the film was damaged by the drop of olive oil or sebum or water, the polymer film is regarded as being non-resistant to olive oil or to sebum.
[0860] Measure of the Resistance Vs Water/Oil and Adhesive Tape can Also be Evaluated
[0861] Mixing of the polymer dissolved in isododecane or isododecane/ethanol with the pigment for 2 minutes at 3500 rpm. The evaluations are performed on BioSkin. In a first stage, a film of each formulation is deposited on a BioSkin sample by means of a film spreader. The thickness of the wet film is 100 μm. The films are dried for 24 hours at room temperature. Once the films are dry, the tests may be performed.
[0862] Resistance to Olive Oil/Sebum
[0863] 0.5 mL of olive oil or sebum is applied to the film of formulation. After 5 minutes, the olive oil or sebum is removed by wiping 15 times with cotton wool. The deterioration of the film following contact with the olive oil or the sebum is thus examined (see
[0864] Resistance to Adhesive Tapes
[0865] A strip of adhesive tape (of Scotch® type) is applied to the film of formulation. A weight is applied to the strip of said tape for 30 seconds. The adhesive tape is then removed and mounted on a slide holder so as to observe the result. The adherence of the film to the support is thus evaluated (see
Example 31: Direct Emulsion Starting with the PHA of Example 24 with a Cationic SA Having an HLB>10
[0866] SA=cationic: cetrimonium chloride
[0867] PHA=(copo PHN at 86% PHHx at 14%)
[0868] 5 g of PHA are dissolved at room temperature with 45 g of ethyl acetate before being dispersed with a solution of 50 g of SA at 1% AM (0.5 g qs=50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm in an ice-water bath at 5° C. for 10 minutes.
[0869] The ethyl acetate is distilled off in successive stages on a bath at 45° C. Evaporation of the water is performed in successive stages on a bath at 45° C. The white dispersion with blueish tints is made up to 35 g with demineralized water.
Example 32: Direct Emulsion Starting with the PHA Described in Example 24 with a Nonionic SA Having an HLB<10
[0870] SA=nonionic: Span 80 (sorbitan monooleate)
[0871] PHA=(copo PHN at 86% PHHx at 14%)
[0872] 5 g of PHA are dissolved at room temperature with 45 g of ethyl acetate before being dispersed with a solution of 50 g of SA at 1% AM (0.5 g qs=50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm in an ice-water bath at 5° C. for 10 minutes.
[0873] The ethyl acetate is distilled off in successive stages on a bath at 45° C. Evaporation of the water is performed in successive stages on a bath at 45° C. The two-phase white suspension is made up to 35 g with demineralized water.
[0874] SA=nonionic: polyglyceryl-4 isostearate
[0875] PHA=(copo PHN at 86% PHHx at 14%)
[0876] 5 g of PHA are dissolved at room temperature with 45 g of ethyl acetate before being dispersed with a solution of 50 g of SA at 1% AM (0.5 g qs=50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm in an ice-water bath at 5° C. for 10 minutes.
[0877] The ethyl acetate is distilled off in successive stages on a bath at 45° C. Evaporation of the water is performed in successive stages on a bath at 45° C. The two-phase white suspension is made up to 35 g with demineralized water.
Example 33: Direct Emulsion Starting with the PHA Described in Example 24 with a Nonionic SA Having an HLB>10
[0878] SA=nonionic: polysorbate 40=Tween 40
[0879] PHA=(copo PHN at 86% PHHx at 14%)
[0880] 5 g of PHA are dissolved at room temperature with 45 g of ethyl acetate before being dispersed with a solution of 50 g of SA at 1% AM (0.5 g qs=50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm in an ice-water bath at 5° C. for 10 minutes.
[0881] The ethyl acetate is distilled off in successive stages on a bath at 45° C. Evaporation of the water is performed in successive stages on a bath at 45° C. The two-phase white suspension is made up to 35 g with demineralized water.
[0882] SA=nonionic: laureth 23
[0883] PHA=(copo PHN at 86% PHHx at 14%)
[0884] 5 g of PHA are dissolved at room temperature with 45 g of ethyl acetate before being dispersed with a solution of 50 g of SA at 1% AM (0.5 g qs=50 g of demineralized water) using an Ultra-Turrax blender (25 mm spindle) at 24 000 rpm in an ice-water bath at 5° C. for 10 minutes.
[0885] The ethyl acetate is distilled off in successive stages on a bath at 45° C. Evaporation of the water is performed in successive stages on a bath at 45° C. The two-phase white suspension is made up to 35 g with demineralized water.
[0886] The stability of the direct emulsions as a function of the nature of the surfactant is summarized in the following table.
TABLE-US-00026 TABLE 22 Stability of the Name of the SA HLB Family direct emulsion Laureth sulfate (SLES) 40 Anionic HLB+ +++ Cetrimoniun chloride 15.8 Cationic HLB+ +++ (CTAC)
[0887] Table 22 summarizes the evaluations for the various emulsions.
TABLE-US-00027 TABLE 22 Examples ex. 26 ex. 27 ex. 28 ex. 31 ex. 29 ex. 30 Emulsion Direct Direct Direct Direct Inverse Inverse direction Water +++ ++ +++ +++ +++ +++ resistance Olive oil +++ +++ +++ ++ ++ ++ resistance Sebum +++ +++ +++ +++ + + resistance Gloss at Matt Matt Matt Matt Matt Matt 20° deposit deposit deposit deposit deposit deposit (gloss = (gloss = (gloss = (gloss = (gloss = (gloss = 5) 6) 5) 7) 5) 7)
[0888] It is seen that the compositions of the invention make it possible to obtain significant resistance to water, oil and sebum.