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(11) | EP 4 512 882 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | A CLEANING COMPOSITION COMPRISING A GLYCOSYL HYDROLASE TARGETING (1,4)-ALPHA-D-GLUCOSIDE BONDS AND AT LEAST ONE GLYCOSYL HYDROLASE TARGETING BETA-GLYCOSIDE BONDS |
| (57) The invention relates to a composition based on enzymes of the class of glycosyl
hydrolases. The given composition can be used as a dishwashing composition, especially
for removal of tough stains and soils based on hemicellulose residues of shells of
cereals, plants and fungi even at lower temperatures.
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FIELD OF THE INVENTION
[0001] The invention relates to a biodegradable detergent composition based on enzymes of the class of glycosyl hydrolases for breakage of alpha- and beta-glycosidic mixed bonds and/or hemicellulose residues in molecules of polysaccharides of cereal crops, starch, cell walls of plants and fungi for prevention of resorption on smooth surfaces. The principle of action of the enzymes consists in catalysis of the reaction of cleavage of beta-1,3-glycosidic, beta-1,4-glycosidic, beta-1,6-glycosidic or alpha-1,4-glycosidic bonds in hemicellulose residues of polysaccharides that consist of monomers of glucose or other monosaccharides and are contained in different parts of plants and the cell wall of fungi. As a result, the composition based on enzymes of the class of glycosyl hydrolases improves removal of tough stains and soils based on hemicellulose residues of shells of cereal crops, plants and fungi from dishware, clothes and other surfaces and prevention of resorption on smooth surfaces at lower temperatures. The composition is intended for inclusion in household dishwashing detergents, including dishwashing powders and gels, dishwasher detergents, including gels and tablets, laundry detergents, including powders, gels and capsules. The composition is biodegradable and can be used for development of household detergents, specifically for creation of dishwasher tablets as well as for dry, liquid and concentrated laundry detergents and liquid dishwashing gels. Use of the detergent composition enables consumers to follow modern nutrition trends due to growth of popularity of high-fiber diets, specifically oats, barley and spelt, superfood-based diets as well as plant-based replacement of dairy products.
BACKGROUND OF THE INVENTION
[0002] At present development of new household detergents with improved consumer properties is one of promising directions. There is a tendency for creation of ecological products for sustainable development purposes. According to statistics of recent years, consumers from different countries started to pay more attention to the composition of products they buy and to check them for presence of non-ecological substances (Novozymes, 2021). By 2025 the demand for products with high contents of natural substances, sustainable products, products free of non-ecological and non-biodegradable substances as well as innovative solutions is expected to grow with the aims of increasing efficiency, naturality and the number of functions of a single product. Consumers prefer additional properties of products and their multifunctionality: environmental friendliness, naturality, safety for pets and children with sensitive skin, usability in homes with self-contained sewage systems and septic tanks, dermatological compatibility and care of sensitive hand skin. The demand for new dishwashing products is also determined by increasing complexity and fixation of soils on smooth surfaces of different functional materials in connection with changing consumers' dietary habits and modern nutritional science trends.
[0003] One of the leading trends in the sphere of packed food products is the healthy lifestyle (Euromonitor International, 2018). This trend resulted in increased consumption of high-fiber products and cereal crops, specifically oats, barley, millet, spelt. The trend for vegetarianism, veganism as well as diagnostics of lactose intolerance defined the need in plant-based alternatives to dairy products. For instance, since the beginning of 2017 the production of oat milk increased four times (Mintel, 2019). This has favorable effects for consumer health; however, high-fiber products can leave tough stains and soils on smooth surfaces.
[0004] Many plant polymers contained in fiber of cereal crops, in different parts of plants and fungi contain alpha-1,4, beta-1,4-, beta-1,3- and beta-1,6-glycosidic mixed bonds and/or hemicellulose residues in grain shells. In connection therewith such compounds have low solubility in water, are firmly fixed on smooth surfaces, sorb other soils and are not easily removed by common detergents as many plant components have adhesive and binding properties leaving tough stains and soils that cannot be removed by common detergents. Examples of such components are an amylose- and amylopectin-based polymer and a polymer based on hemicellulose residues of saccharides. At present household detergents used for removal of such soils from dishware, cutlery and clothes mainly include alpha-amylase, a natural enzyme accelerating cleavage of (1,4)-alpha-D-glucosidic bonds in starch molecules. Starch soils are firmly fixed on surfaces of different fabrics, including delicate fabrics, and it is difficult to remove them in low-temperature wash cycles. In addition, starch soils make laundry stiff, which can be felt by the skin surface during daily wear. Nevertheless, alpha-amylase can cleave (1,4)-alpha-D-glucosidic bonds in starch only. In connection therewith alpha-amylase can be used as a supplemental component for removal of starch from different surfaces, however it has no ability to cleave hemicellulose residues and beta-D-glucosidic bonds in cell walls of plants and fungi.
[0005] A high content of polysaccharides in cereal crops, cell walls of plants and fungi is made up by β-D-glucose polysaccharides consisting of glucose links and connected with (1,3)-beta-, (1,4)-beta and (1,6)-beta-D-glucosidic bonds. This peculiarity explains their low solubility in water. Such bonds are also present in cell walls of other plants - arabinoxylans (pentosans) consisting of copolymers of two pentose sugars: arabinose and xylose, the bonds between which can also be broken by glucan-hydrolases. The content of arabinoxylans is the highest in shells of cereal crops (30-50%) and much lower (2.5-4.5%) in grain endosperm. Pentosans of plant origin are insoluble in water and are firmly fixed on different smooth surfaces of metal, glass and plastic. Thus, removal of soils based on cellulose and/or hemicellulose residues during dishwashing and laundering is a recognized problem and there is considerable interest in search for effective combinations that can decompose polysaccharides of D-glucose monomers connected with different D-glucosidic bonds that are contained therein.
[0006] Glucosyl hydrolases (E.C. 3.2.1 - x) are a class of enzymes catalyzing hydrolysis of O- or S-glycosides. Glucosyl hydrolases can also be classified as exo- or endo-acting, depending on the fact whether they act on the terminal or internal monosaccharide residue in the hydrocarbon chain of cereals, other plants and in the cell wall of fungi, respectively. The class of glucosyl hydrolases includes licheninase (EC 3.2.1.73), which is capable of breaking shells of cereal crops and preventing resorption thanks to hydrolysis by (1,4)-beta-D-glucoside bonds in mixed hemicellulose residues in cell walls of plants and fungi. Laminarinase (EC 3.2.1.6) catalyses cleavage of internal (1,3)-beta-D-glucosidic and (1,4)-beta-D-glucosidic bonds in mixed hemicellulose residues in cell walls of plants and fungi. β-glucosidases (EC 3.2.1.21) are important representatives of a large class of glycosyl hydrolases. They contribute to complete dissolution and removal of hemicellulose residues by hydrolysis of β-glucosidic bonds present either in disaccharides, oligosaccharides or in so called conjugated glucosides such as coniferin and syringin. Beta-glucanase is an important lyase widely distributed in bacteria, fungi, plants and marine animals and it is subdivided into exo-β-1,3-glucanase (EC3.2.1.58) and endo-β-1,3-glucanase (EC3.2.1.6 or EC3.2.1.39). Exo-β-1,3-glucanase successively cuts glucose residues at the non-reducing end, which mainly results in glucose. Endo-β-1,3-glucanase randomly cleaves β-1-3-glycosidic bonds along the β-glucan chain and the final product is mainly glucan oligosaccharide. Another representative of the class of glucosyl hydrolases is alpha-amylase (E.C. 3.2.1.1), which cleaves (1,4)-alpha-D-glucosidic bond of starch and other related polysaccharides with formation of simple sugars such as glucose, maltose and limit dextrin.
[0007] At present glucosyl hydrolases isolated from mesophilic bacteria are used. Main problems of glucosyl hydrolases now in use are low activity and instability at high temperatures. It is known that alpha-amylase activity is optimal within the pH range of 6.0-7.5 in the temperature interval of 24-32°C, while laminarinase activity is optimal within the pH range of 5.5-6.5 in the temperature interval of 24-32°C [Temperature dependency of carbohydrase activity in the hepatopancreas of thirteen estuarine and coastal bivalve species from the North American east coast / Vibeke Brock, Victor S. Kennedy, Axel Brock/ J. Exp. Mar. Biol. Ecol., 1986, Vol. 103, pp. 87-101 Elsevier https://doi.org/10.1016/0022-0981(86)90134-6]. It is also known that β-glucosidase activity is optimal at 25°C and pH 6.5 [Enzyme and Microbial Technology / Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family from a psychrotolerant bacterium Micrococcus antarcticus / Hong-Xia Fan, Li-Li Miao, Ying Liu, Hong-Can Liu, Zhi-Pei Liu / Volume 49, Issue 1, 10 June 2011, Pages 94-99 https://doi.org/10.1016/j.enzmictec.2011.03.001], while laminarinase maintains its maximum activity at 45°C and pH 4.5-5.5 and loses its 45% activity in extreme acidic and basic conditions (pH 3.0 and 11.0) [Characterisation of a novel laminarinase from Microbulbifer sp. ALW1 and the antioxidant activity of its hydrolysates Qingsong Hu, Xiaoqian Yin, Hebin Li, Xinghua Wang, Zedong Jiang, Lijun Li, Hui Ni, Qingbiao Li, Yanbing Zhu / International Journal of Food Science and Technology 2021 https://doi.org/10.1111/ijfs.15041].
[0008] Alpha-amylase is one of the most frequently used enzymes in household detergents as it is capable of breaking complex hydrocarbons and polysaccharides, which makes it an irreplaceable ingredient in production of washing and cleaning products. However, there are restrictions in use of this enzyme in household detergents. First of all, use of alpha-amylase can cause damage of fabrics and reduction in quality of textile articles [Fernandes, P., Sadocco, P., & Alonso-Varona, A. (2017). Enzymes in the cleaning industry: An overview of current trends and technological improvements. Critical reviews in biotechnology, 37(5), 609-620]. Besides, the enzyme can be ineffective when used in aquatic solutions with low pH, specifically pH below 7.0 [Tiwari, R., Nain, L., & Singh, V. (2015). Alpha-amylase immobilization on polyaniline coated magnetic nanoparticle: a novel approach for biocatalysis in acidic medium. Bioprocess and biosystems engineering, 38(3), 463-472]. Specifically, alpha-amylase activity decreases by more than 36% at pH 6.0 [Carvalho RV, Côrrea TL, da Silva JC, de Oliveira Mansur LR, Martins ML. Properties of an amylase from thermophilic Bacillus SP. Braz J Microbiol. 2008 Jan;39(1):102-7.]. It is also known that use of the enzyme in high concentrations can cause problems with safety and health of consumers of household detergents [Zhao, J., Li, C., & Wang, X. (2017). Production, purification, and application of α-amylase from Bacillus subtilis in detergent industry. Brazilian Journal of Microbiology, 48(1), 31-39]. Therefore, it is necessary to thoroughly monitor the enzyme concentration when it is used in household detergents. On the whole, alpha-amylase is an important ingredient in household detergents, but it is necessary to take into account the above mentioned restrictions of its use. The authors surprisingly discovered that the composition based on glucosyl hydrolase and alpha-amylase maintains high activity within the basic pH range of 10.0-12.5, which enables its introduction in basic powder laundry detergents, surface cleaners and dishwasher tablets.
[0009] The authors surprisingly discovered that the composition based on glucosyl hydrolases maintains its activity within the pH range of 10-12, which is optimal for most detergents. It was shown that presence of exo-glucosyl hydrolase does not reduce effectiveness of endo-glucosyl hydrolase with respect to stains or soils based on firmly fixed mixed hemicellulose residues in cell walls of plants and fungi, but on the opposite increases it and demonstrates synergism for a significant rise in performance of removal of stains and soils even at temperatures within the range of 30 to 60°C. This temperature range is usually used in ecocycles of washing machines and dishwashers and is also optimal for hand dishwashing and laundering. Thus, high activity of enzymes at low temperatures is in line with eco-trends and convenient use of household detergents, which enables achieving effective removal of mixed soils and preventing resorption on smooth surfaces.
[0010] The document WO2018060216A1 published on 05.04.2018 describes the ingredients of a detergent composition for removal and/or reduction of soils on surfaces and for dishwashing that include alpha-amylase, protease and can include other enzymes, including licheninase. The above described composition has a number of disadvantages that are described below. The patent application specifies high temperatures for implementation of the detergent composition, namely 50-95°C. Such temperature is suitable for use in a dishwasher, in industrial cleaning, but it is too high for use in hand dishwashing or laundering as this temperature is uncomfortable for hands and can damage delicate fabrics such as wool, silk, cashmere. There are data which state that enzymes of the class of glucosyl hydrolases maintain only 20% of their activity at temperatures from 60 to 80 °C [Joshi, J.B., Priyadharshini, R. & Uthandi, S. Glycosyl hydrolase 11 (xynA) gene with xylanase activity from thermophilic bacteria isolated from thermal springs. Microb Cell Fact 21, 62 (2022). https://doi.org/10.1186/s12934-022-01788-3]. It is also mentioned in the document that surfactants can be used in the composition; however it was shown that alpha-amylase and proteases can lose their activity in presence of surfactants both at room and higher wash cycle temperatures [Zhang, J., Zhang, J. Study on the interaction of alkaline protease with main surfactants in detergent. Colloid Polym Sci 294, 247-255 (2016). https://doi.org/10.1007/s00396-015-3777-3; Lund, H., Kaasgaard, S.G., Skagerlind, P. et al. Correlation Between Enzyme Activity and Stability of a Protease, an Alpha-Amylase and a Lipase in a Simplified Liquid Laundry Detergent System, Determined by Differential Scanning Calorimetry. J SurfactDeterg 15, 9-21 (2012). https://doi.org/10.1007/s11743-011-1272-5; O. Herrera-Marquez, M. Fernández-Serrano, M. Pilamala, M.B. Jácome, G. Luzón, Stability studies of an amylase and a protease for cleaning processes in the food industry, Food and Bioproducts Processing, 2019, https://doi.org/10.1016/j.fbp.2019.06.015]. It is also known that use of proteases in combination with glucosyl hydrolases in laundry detergents for delicate fabrics inhibits hydrolases suppressing their activity [Daniela J. Sueldo, Alice Godson, Farnusch Kaschani, Daniel Krahn, Till Kessenbrock, Pierre Buscaill, Christopher J. Schofield, Markus Kaiser, Renier A. L. van der Hoorn. Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface. New Phytologist, 2023. https://doi.orh/10.1101/2022.12.12.520059]. The patent application also mentions use of chelating agents, however it should be noted that chelating agents such as DTPA and EDTA were prohibited in some countries, for example, in Sweden and Norway because of destructive effects of the chemical substances on aquatic species and contamination of sewage waters. In addition, the patent application includes data about use of a protease inhibitor for improvement of stability of the composition, but this can reduce protease activity. The authors of the invention use boric acid derivatives as inhibitors that are prohibited by ecological certificates because of non-biodegradability. As a result, this composition cannot be ecologically safe and cannot obtain voluntary ecological product certification.
The international patent application WO2017129754A1 published on 03.08.2017 suggests use of beta-glucanase or licheninase variants (with amino acid replacements) in combination with amylase in detergent compositions for cleaning, laundering and dishwashing for removal of beta-glucans. However, the described composition has a number of disadvantages. It is stated in the document that pH for the composition is 5.5-13.5. Such a range causes a number of difficulties connected with stability of enzymes, in particular beta-glucanases, due to the following reasons: 1) stability of beta-glucanases decreases in liquid detergents with high pH; 2) beta-glucanase derived from Bacilus amyloliquefaciens and Bacillus subtilis has optimum pH=6, while at pH=10 its activity is as low as 1-11%. In one of the embodiments the composition of the invention comprises one or several amylases and the given composition variant has a synergistic effect. However, this REM synergistic effect is over 6.5 at 40°C during 30 minutes at pH=7.5, at the more basic value pH=10 the REM synergistic effect decreases and makes up only 6.1. Also the invention is selective as it acts only on beta-1,3 and beta-1,4 and in this case the detergent will be effective only against individual groups of polysaccharides and will not be a universal detergent since it does not act on beta-1,6- and alpha-glycosidic bonds that are present in different food plant components. It is also suggested to use alpha-amylase as an additional active component, however there are literature data that alpha-amylase activity can be inhibited by beta-glucan destruction products [Gong L, Feng D, Wang T, Ren Y, Liu Y, Wang J. Inhibitors of α-amylase and α-glucosidase: Potential linkage for whole cereal foods on prevention of hyperglycemia. Food Sci Nutr. 2020 Nov 4;8(12):6320-6337. doi: 10.1002/fsn3.1987]. This fact can be a restriction for use of the composition as the components will self-inhibit in case of co-presence in household detergents. The patent states that the composition can display beta-glucanase activity during 15 minutes, but the more preferential processing duration is min. 60-90-120 minutes. The given data do not make it possible to conclude whether the composition will be effective in case of a short duration of its use in case of hand dishwashing, which on average lasts 1-10 minutes. It is also specified in the document that use of anionic surfactants in the composition is preferential, however, as it was stated in the review of the previous document, anionic surfactants can inactivate amylase [Zhang, J., Zhang, J. Study on the interaction of alkaline protease with main surfactants in detergent. Colloid Polym Sci 294, 247-255 (2016). https://doi.org/10.1007/s00396-015-3777-3; Lund, H., Kaasgaard, S.G., Skagerlind, P. et al. Correlation Between Enzyme Activity and Stability of a Protease, an Alpha-Amylase and a Lipase in a Simplified Liquid Laundry Detergent System, Determined by Differential Scanning Calorimetry. J Surfact Deterg 15, 9-21 (2012). https://doi.org/10.1007/s11743-011-1272-5]. The document also mentions use of glycerin and propylene glycol, which are known anti-foaming agents and can have a negative effect on properties of a detergent composition in the form of dishwasher powder or tablets.
[0011] The document WO1995031533A1 published on 23.11.1995 describes an enzyme with endo-beta-glucanase activity for addition in different compositions with the aim of degradation or modification of beta-glucan-containing materials. The authors of the document suggested several variants of enzyme use: 1) for destruction or lysing of cell walls of microorganisms, thus enabling extraction of desired products produced by the microorganism, 2) for extraction of mannan protein from the external layer of yeast cell walls, 3) for production of protoplasts from fungi or yeast, 4) for brewing and production of grape wine or squeezed juice, 5) for improvement of quality of bakery products and other cereal products, 6) for extraction of aromatic compounds from plant raw materials, 7) as an active ingredient in cleaning compositions for dental prostheses and mouthwashes, 8) for removal of biofilms from lens boxes, 9) for removal of excess dyes from textile articles. The use of the enzyme in detergent compositions is not provided for and is not disclosed in the invention. Besides, one of variants of use is removal of excess dyes, therefore use of this enzyme in laundry detergents is restricted - the enzyme can cause textile color fading, however this use does not encompass inclusion in dishwasher detergents.
SUMMARY OF THE INVENTION
[0012] In the first aspect the invention relates to a composition intended for use in household detergents for automatic and/or hand washing of dishware made of different functional materials, which are active at pH 10.0-12.5 and water hardness of 0-42 dH and consist of:
(A) Endo glucosyl hydrolase for degradation of alpha-D-glucosidic bonds in mixed polysaccharides, specifically alpha-amylase obtained biotechnologically from yeast-like fungi or microorganisms resistant to protease presence, where the specified alpha-amylase has biological activity over 100 U/g, pH 4-10 at 25°C, viscosity 0-1000 sPas at 25°C;
(
) Exo or endo glucosyl hydrolase for degradation of beta-D-glucosidic bonds in mixed
polysaccharides selected from beta-glucanase, beta-glucosidase, cellulase or laminarinase
obtained biotechnologically from microorganisms, with activity over 100 U/g, pH 4-10
at 25°C, viscosity 0-1000 sPas at 25°C;
where the mass ratio of components A and B is (0.0025-0.25):(0.0010-0.25), respectively.
[0013] In the second aspect the invention relates to use of the composition of the invention in a detergent for automatic and/or hand dishwashing. The detergent of the invention can contain 0.0025-0.50% wt. of the composition of the invention.
[0014] Acceptable auxiliary substances in the detergent of the invention can be selected from the following categories of components.
Anionic surfactants:
[0015] Salts of higher carboxylic acids with the general formula: R1-CO2X1, where R1 is an alkyl and / or alkenyl group with a long hydrocarbon chain from 5 to 21 atoms unexpectedly, and X1 is an alkali and/or alkaline earth metal, ammonium, alkylammonium, alkanolammonium, glucoammonium cation, basic amino acids;
[0016] Alkyl polyethylene glycol sulfate with the general formula R2-O(-CH2-CH2-O)n1 (SO3) n2 X1, wherein n1 takes a value from 0 to 10 and denotes the number of polyethylene groups, R2 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms, X1 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium, basic amino acid;
[0017] Alkyl sulfate with the general formula R3-OSO3X3, where R3 is an alkyl and / or alkenyl group with a long hydrocarbon chain from 6 to 22 carbon atoms, and X3 is an alkali and/or alkaline earth metal, ammonium, alkylammonium, alkanolammonium, glucoammonium cation;
[0018] Amide salt of a higher fatty acid and methylglycine with the general formula R4-C(O)-N(-CH3)-CH2-CO2X4, wherein R4 is an alkyl and/or alkenyl group with a hydrocarbon chain length from 5 to 21 carbon atoms and X4 is an alkali and/or alkaline earth metal cation, ammonium, alkanolammonium, glucoammonium;
[0019] An alkyl polyethylene glycol carboxylate with the general formula: R5-O(-CH2-CH2-O-)n2CH2-CO2X5, wherein n2 can take values from 1 to 15 and denotes the number of polyethylene glycol groups, R5 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms and X5 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0020] A bisubstituted salt of 2-sulfo carboxylic acid with the general formula: R6-CH(-SO3X6)-CO2X6, wherein R6 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 4 to 20 carbon atoms and X6 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0021] A mono- or divalent amide salt of a higher carboxylic acid and glutamic acid with the general formula: R7-C(O)-NH-CH(-CH2-CH2-CO2X7)-CO2X7, wherein R7 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and X7 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanoammonium, glucoammonium or hydrogen;
[0022] An amide salt of a higher fatty acid and glycine with the general formula: R8-C(O)-NH-CH2-CO2X8, wherein R8 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, and X8 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0023] An amide salt of a higher fatty acid and alanine with the general formula: R9-C(O)-NH-CH(-CH3)-CO2X9, wherein R9 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and X9 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0024] An amide salt of a higher fatty acid and 2-aminomethylethanesulfonic acid with the general formula: R10-C(O)-N(-CH3)-CH2-CH2-SO3X10, wherein R10 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, and X10 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0025] An alkylpolyglucoside hydroxypropyl sulfonate with the general formula: R11-O-[G]p1-O-CH2-CH(-OH)-CH2-SO3X11, wherein R11 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms, G is a saccharide fragment containing 5 or 6 carbon atoms, p1 can take values from 1 to 4, and X11 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0026] An alkylpolyglucoside carboxylate with the general formula: R12-O-[G]p2-O-CH2-CO2X12, wherein R12 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms, G is a saccharide fragment containing 5 or 6 carbon atoms, p2 can take values from 1 to 4, and X12 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0027] An amide salt of a higher fatty acid and a threonine with the general formula: R13-C(O)-NH-CH(-CH(-OH)-CH3)-CO2X13, wherein R13 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, and X13 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0028] An amide salt of a higher fatty acid and an amino acid obtained by hydrolysis of proteins from vegetable raw materials, with the general formula: R14-C(O)-AAX14, wherein R14 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, AA is an amino acid or peptide obtained by hydrolysis of plant protein (possible protein sources: apple, soybean, wheat, cotton etc.), and X14 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium.
Amphoteric surfactants:
[0029] A divalent salt of acylamphodiacetate with the general formula: R15-C(O)-NH-CH2-CH2-N(-CH2-CO2X15)-CH2-CH2-O-CH2-CO2X15, wherein R15 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and X15 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0030] An acylamphoacetate salt with the general formula: R16-C(O)-NH-CH2-CH2-N(-CH2-CO2X16)-CH2-CH2-OH, wherein R16 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and X16 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0031] An alkylamphoacetate salt with the general formula: R17-C(=N-CH2-CH2-N((-CH2-CH2-OH)-CH2-CO2X17)-), wherein R17 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and X17 is an alkali and/or alkaline earth metal cation, ammonium, alkylammonium, alkanolammonium, glucoammonium;
[0032] An acylamidoalkylbetaine with the general formula: R18-C(O)-NH-R19-N(-CH3)2)-CH2-CO2, wherein R18 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, R19 is an alkyl group with a hydrocarbon chain length of 1 to 4 carbon atoms;
[0033] An acylamidoalkylhydroxysultaine with the general formula: R20-C(O)-NH-R21-N(-CH3)2-CH2-CH(-OH)-CH2-SO3, wherein R20 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, R21 is an alkyl group with a hydrocarbon chain length of 1 to 4 carbon atoms;
[0034] An acylamidoalkylamine oxide with the general formula: R22-C(O)-NH-R23-N(-CH3)2-O, wherein R22 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms, R23 is an alkyl group with a hydrocarbon chain length of 1 to 4 carbon atoms;
[0035] An alkylbetaine with the general formula: R24-N(-CH3)2)-CH2-CO2, wherein R24 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms;
[0036] An alkylhydroxysultaine with the general formula: R25-N(-CH3)2-CH2-CH(-OH)-CH2-SO3, wherein R25 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms;
[0037] An alkylsultaine with the general formula: R26-N(-CH3)2-CH2-CH2-CH2-SO3, wherein R26 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms;
[0038] An alkylamine oxide with the general formula: R27-N(-CH3)2-O, wherein R26 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms.
Nonionic surfactants:
[0039] An alkyl glucoside with the general formula: R28-O-[G]p3, wherein R28 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 4 to 22 carbon atoms, G is a saccharide fragment containing 5 or 6 carbon atoms, p3 can take values from 1 to 4;
[0040] An alkylpolyethylene glycol with the general formula: R29-O(-CH2-CH2-O-)n3H, wherein n3 can take values from 2 to 20, and denotes the number of polyethylene glycol groups, R29 being an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms;
[0041] An alkylpolyethylene/propylene glycol with the general formula: R30-O(-CH2-CH2-O-)n4(-CH(-CH3)-CH2-O-)n5H, wherein n4 can take values from 2 to 20 and denotes the number of polyethylene glycol groups, n5 can take values from 2 to 20 and denotes the number of polypropylene glycol groups, R30 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms;
[0042] A dialkyl polyethylene glycol with the general formula: R31-O(-CH2-CH2-O-)n6R32, wherein n6 can take values from 2 to 20, and denotes the number of polyethylene glycol groups, R31 being an alkyl and/or alkenyl group with a hydrocarbon chain length of 6 to 22 carbon atoms, R32 being an alkyl and/or alkenyl group with a hydrocarbon chain length of 1 to 12 carbon atoms;
[0043] A dialkylpolyethylene/propylene glycol with the general formula: R33-O(-CH2-CH2-O-)n7(-CH(-CH3)-CH2-O-)n8-R34, wherein n7 can take values from 2 to 20, and denotes the number of polyethylene glycol groups, n8 can take values from 2 to 20, and denotes the number of polypropylene glycol groups, R33 is an alkyl and/or alkenyl group with a hydrocarbon chain length from 6 to 22 carbon atoms, R34 is an alkyl and/or alkenyl group with a hydrocarbon chain length from 1 to 12 carbon atoms.
Dispersing medium for the polysaccharide/solvent:
[0044] An organic alcohol with the general formula: R35(-OH)s1, wherein R35 is an alkyl group with a hydrocarbon chain length from 3 to 12 carbon atoms, s1 can take values from 1 to 12, and denotes the number of hydroxyl groups arranged randomly in a hydrocarbon radical relative to each other;
[0045] An alkylpolypropylene glycol with the general formula: H(-CH(-CH3)-CH2-O-)n9R36, wherein n9 can take values from 2 to 10 and denotes the number of polypropylene glycol groups, R36 is an alkyl group with a hydrocarbon chain length from 1 to 10 carbon atoms.
[0046] Polysaccharides: cellulose gum and derivatives, cyclodextrines of different forms and modifications, particularly beta-cyclodextrines.
pH regulators:
[0047] Organic acids with the general formula: R37(-OH)s2(-COOH)m1, wherein R37 is an alkyl group with a hydrocarbon chain length of 1 to 12 carbon atoms, s2 can take values from 1 to 12 and denotes the number of hydroxyl groups arranged in the hydrocarbon radical in random order with respect to each other, m1 can take values from 1 to 4 and denotes the number of carboxyl groups arranged in the hydrocarbon radical in random order with respect to each other;
[0048] Solutions of alkali or alkaline earth metal hydroxides, ammonia, primary and tertiary alkylamines, primary and tertiary alkanolamines, primary and tertiary glucamines, basic amino acids, disodium salt of citric acid, trisodium salt of citric acid.
Inhibitors of the reverse sedimentation of contamination:
[0049] Polysaccharide derivatives: sodium salt of carboxymethyl polysaccharide, hydroxyalkyl polysaccharide, alkyl polysaccharide and cellulose gum;
[0050] Polyvinylpyrrolidone and its derivatives, copolymers of polyvinylpyrrolidone and vinylimidazole;
[0051] Water soluble salts of polyacrylic acid, polymethacrylic acid, copolymers of acrylic/methacrylic and maleic acid.
Defoamers:
[0052] Higher carboxylic acids with the general formula: R39-CO2H, wherein R39 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms;
[0053] Higher carboxylic alcohols with the general formula: R40-COH, wherein R40 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms;
[0054] Esters of higher carboxylic alcohols with the general formula: R41-O-R42, wherein R41, R42 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 4 to 22 carbon atoms;
[0055] Bisamides of alkyl diamines and higher carboxylic acids with the general formula: R43-C(O)-NH-R44-NH-C(O)-R45, wherein R43, R45 is an alkyl and/or alkenyl group with a hydrocarbon chain length of 5 to 21 carbon atoms and R44 an alkyl radical with a hydrocarbon chain length of 1 to 12 carbon atoms;
[0056] Builders and fillers: sodium silicate, sodium sulfate, sodium chloride, sodium carbonate and other commercially available components.
[0058] Complexing agents: phosphonates of any forms, methylglycine N,N-diacetic acid, trisodium salt, L-glutamic acid N,N-diacetic acid tetra sodium salt, polycarboxylates,
[0059] Enzymes: protease (ficin, bacillolysin, subtilisin), alpha-amylase, pectate lyase, mannanase, mannosidase, cellulase, amino oxidase, feruloyl esterase, beta-glucanase, tannase, alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, manganese peroxidase, licheninase, xylanase and other commercially available enzymes used in laundry, dishwashing, floor, glass and all-purpose cleaners;
[0060] Bleaching agents based on oxygen compounds: hydrogen peroxide, calcium peroxide, carbamide peroxide, ε-phthalimidoperoxycaproic acid, sodium carbonate peroxide, TAED, manganese salts or complexes and other commercially available components;
[0061] Preservatives: organic acids and salts of alkali and alkaline earth metals, ammonium, alkylammonium, alkanolammonium, glucoammonium corresponding to these acids: benzoic acid, sorbic acid, 4-methoxybenzoic acid, salicylic acid, undecylenic acid;
[0062] Organic alcohols and phenols: phenoxyethanol, benzyl alcohol, caprylyl glycol, ethylhexyl glycerolrine, phenethyl alcohol, 3-methyl-4-isopropyl phenol, 2,4-dichlorobenzyl alcohol;
[0063] Broad spectrum biocides: benzisothiazolinone, dodecyldipropylene triamine, methylisothiazolinone, mixture of these, sodium pyrithione, laurylamine dipropylenediamine and other commercially available components;
[0065] Essential oil fragrances or essential oils in pure form or as blends in various proportions: orange, bergamot, lemon, lime, mandarin, grapefruit, neroli, rosewood, yuzu, lemongrass, lavender, sage, thyme, melissa, mint, tea tree, eucalyptus, cedar, sandalwood, black pepper, pink pepper, cinnamon, cardamom, coriander, jasmine, rose, peony, blue chamomile or other available essential oil;
Different pigments or dyes of synthetic or natural origin.
[0066] In another aspect the invention relates to a biodegradable detergent composition based on enzymes of the class of glycosyl hydrolases for destruction of alpha- and beta-glycosidic mixed bonds and/or hemicellulose residues in molecules of polysaccharides of cereal crops, starch, cell walls of plants and fungi for prevention of resorption on smooth surfaces. The composition based on enzymes of the class of glycosyl hydrolases improves removal of tough stains and soils based on hemicellulose residues of shells of cereal crops, plants and fungi from dishware, clothes and other surfaces and prevention of resorption on smooth surfaces at lower temperatures. Use can be different in that the composition can be used for development of detergents, specifically for creation of dry dishwasher tablets as well as for dry, liquid and concentrated laundry detergents and dishwashing gels.
EXPERIMENTAL PART
[0067] The examples included in the present description are not limiting with respect to the claimed invention and are given with the aim of illustration and proof of achievement of expected technical results only. These examples are ones of many experimental data obtained by the authors of the invention that support efficiency of detergents within the scope of the invention.
Example 1.
[0068] The components to be included in the composition of the invention were studied as part of different dishwashing detergents. A liquid detergent (formula No.1, formula No.2), specifically a universal dishwashing liquid, within the scope of the invention was prepared (Table 1, Table 2).
Table 1. Ingredients of a basic concentrated foam dishwashing liquid
| No. | Component | Content, % wt. |
| 1 | Purified water | up to 100.00 |
| 2 | Combination of surfactants: sodium laureth sulfate 70% solution, cocamidopropyl betaine 40-45% solution, decyl glucoside 50-53% solution | 0.5-30.5 |
| 3 | Food grade sodium chloride | 1.2-6.2 |
| 4 | Trisodium citrate dihydrate | 0.01-0.6 |
| 5 | Citric acid monohydrate | 0.05-0.25 |
| 6 | Combined preservative based on BIT, sodium pyrithione and BDA | 0.005-0.2 |
| 7 | Aqueous solution of a citric acid salt and silver citrate | 0-0.1 |
| 8 | Botanical cotton extract | 0.01-0.15 |
| 9 | Aromatic composition based on lemongrass essential oil | 0.0-1.0 |
[0069] For preparation of the final detergent of the invention it is necessary to add composition components: 0.0025-0.05% alpha-amylase, 0.001-0.10% enzyme of the class of glucanohydrolases, for example, β-1,3-1,4-glucanase, β-1,3-1,4-1,6-glucanase, licheninase, β-glucosidase, cellulase.
Table 2. Ingredients of a basic concentrated dishwashing gel
| No. | Component | Content, % wt. |
| 1 | Purified water | up to 100.00 |
| 2 | Composition based on surfactants: decyl glucoside 50-53% solution, cocamidopropyl betaine 40-45% solution, sodium cocoamphoacetate 30-32% solution, sodium cocoylglutamate, sodium coco-sulfate | 0.5-33.5 |
| 5 | Food grade sodium chloride | 0.5-2.1 |
| 6 | Trisodium citrate dihydrate | 0.01-0.6 |
| 7 | Citric acid monohydrate | 0.01-0.5 |
| 8 | Preservative benzyl alcohol | 0.005-0.6 |
| 9 | Aqueous solution of a citric acid salt and silver citrate | 0-0.02 |
| 10 | Botanical cotton extract | 0.05-0.15 |
| 11 | Aromatic composition based on tangerine essential oil | 0.0-1.0 |
[0070] For preparation of the final detergent of the invention it is necessary to add composition components: 0.0025-0.05% alpha-amylase, 0.001-0.10% enzyme of the class of glucanohydrolases, for example, β-1,3-1,4-glucanase, β-1,3-1,4-1,6-glucanase, licheninase, β-glucosidase, cellulase. The prepared dishwashing liquid provides for high efficiency of cleaving beta-1,3-glycosidic, beta-1,4-glycosidic, beta-1,6-glycosidic or alpha-1,4-glycosidic bonds in hemicellulose residues of polysaccharides present either in disaccharides, oligosaccharides or in so called conjugated glucosides, such as coniferin and syringin, at any running water hardness 0-42 dH, at any dishwashing temperature from +10°C to +45°C and at pH=7.5-8.5. It does not leave limescale or streaks, is fully washed from dishware and glass surfaces, suits for washing dishware of children and people with sensitive hand skin, is stable during storage within 24 months (observation period).
Example 2.
[0071] The components to be included in the composition of the invention were studied as part of different detergents. A dry detergent, specifically, tableted automatic dishwasher powder, within the scope of the invention was prepared (Table 3).
Table 3. Ingredients of basic automatic dishwasher tablets
| No. | Component | Content, % wt. |
| 1 | Sodium carbonate | 15.5-35.5 |
| 2 | Sodium gluconate | 13.8-20.8 |
| 3 | Oxygen bleaching agent of the class of bleaching agents, specifically sodium percarbonate | 9.5-25.5 |
| 4 | Anhydrous citric acid or monohydrate or dihydrate | 2.5-7.0 |
| 5 | Methylglycinediacetic acid trisodium salt | 2.0-7.0 |
| 6 | Bleaching agent activator of the class of activators, specifically TAED or manganese salt | 0.5-4.2 |
| 7 | Baking soda, namely sodium hydrocarbonate | 0.5-3.5 |
| 8 | Non-ionic surfactant, namely fatty alcohol alkoxylate made from a C12-C14- fatty alcohol | 0.25-4.5 |
| 9 | Protease (subtilisin) | 0.0025-0.25 |
| 10 | Aromatic composition based on eucalyptus or bergamot and yuzu essential oil | 0.0-1.0 |
| 11 | PEG-4000 | 0.01-2.0 |
| 12 | Polymeric dispersing agent of the group of acrylates, specifically polyacrylic acid, Na-salt, partly neutralized | 0.01-10.0 |
| 13 | Polycarboxylates | 0.0-3.0 |
| 14 | Dye or pigment or a coloring mixture | 0.0-1.0 |
| 15 | Functional additives (if required) | 0.0-5.0 |
[0072] For preparation of the final detergent of the invention it is necessary to add composition components: 0.0025-0.05% alpha-amylase, 0.001-0.10% enzyme of the class of glucanohydrolases, for example, β-1,3-1,4-glucanase, β-1,3-1,4-1,6-glucanase, licheninase, β-glucosidase, cellulase. The prepared dry dishwasher powder provides for high efficiency of cleavage of beta-1,3-glycosidic, beta-1,4-glycosidic, beta-1,6-glycosidic or alpha-1,4-glycosidic bonds in hemicellulose residues of polysaccharides present either in disaccharides, oligosaccharides or in so called conjugated glucosides, such as coniferin and syringin, at any running water hardness 0-42 dH and at any dishwashing temperature from +40°C to +65°C, at pH=10.5-12.5. It does not leave limescale, streaks, drops and water stains, does not change glass properties and protects dishwashers prolonging their service life. It does not leave odor on dishware after washing. It is suitable for all materials (glass, metal, plastic, teflon, etc.) and is stable during storage within 24 months (observation period).
Example 3.
[0073] The components to be included in the composition of the invention were studied as part of different cleaners for different surfaces. A liquid detergent (pH=7-8), specifically a universal cleaner for kitchen surfaces, within the scope of the invention was prepared (Table 4).
Table 4. Ingredients of a basic universal spray for different surfaces
| No. | Component | Content, % wt. |
| 1 | Purified water | up to 100 |
| 2 | Composition based on surfactants: decyl glucoside 50% solution, potassium cocoate 43% solution | 0.05-10.5 |
| 3 | Chelating agent, specifically glutamic acid, N,N-diacetic acid, tetra sodium salt | 0.05-0.5 |
| 4 | Citric acid monohydrate | 0.05-0.25 |
| 5 | Combined preservative based on BIT, sodium pyrithione and BDA | 0.005-0.2 |
| 6 | Botanical cotton Extract | 0.01-0.15 |
| 7 | Aqueous solution of a citric acid salt and silver citrate | 0-0.02 |
[0074] For preparation of the final detergent of the invention it is necessary to add composition components: 0.0025-0.05% alpha-amylase, 0.001-0.10% enzyme of the class of glucanohydrolases, for example, β-1,3-1,4-glucanase, β-1,3-1,4-1,6-glucanase, licheninase, β-glucosidase, cellulase. The prepared universal cleaner for different kitchen surfaces and utensils provides for high efficiency of cleavage of beta-1,3-glycosidic, beta-1,4-glycosidic, beta-1,6-glycosidic or alpha-1,4-glycosidic bonds in hemicellulose residues of polysaccharides present either in disaccharides, oligosaccharides or in so called conjugated glucosides, such as coniferin and syringin, at any running water hardness 0-42 dH at any dishwashing temperature from +10°C to +35°C, at pH=7-8. It does not leave streaks, limescale, drops and water stains, does not change properties of benchtops and furniture, home appliances, cooktops, kitchen sinks, pans, frying pans and sheet pans. It is easily washable and gives a shine. It neutralizes unpleasant odors and has an aroma effect. It is suitable for all materials (glass, metal, plastic, wood, teflon) and is stable during storage within 24 months (observation period).
Example 4.
[0075] A study was carried out to determine activity of composition enzymes of the class of glycosyl hydrolases for breakage of alpha- and beta-glucosidic bonds and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans. The study was intended to test the hypothesis about breakage of complex mixed polysaccharides with different glucosidic bonds and synergism of enzymes of the class of glucosyl hydrolases.
[0076] The analysis methods were based on stage-by-stage enzymatic hydrolysis of (1→3)(1→4)(1→6)-β-D-glucans from different sources of plant or biotechnological origin with the use of enzymes of the class of glucosyl hydrolases of the invention to β-glucooligosaccharides and glucose, respectively. The mass ratio of hydrolysed (1→3)(1→4)(1→6)-β-D-glucans is determined by the colorimetric method by the coloring degree of glucose molecules with the glucose oxidase reagent, which speaks of activity of enzymes. To evaluate activity of enzymes of the class of glucosyl hydrolases and correspondingly efficiency of degradation of α- and β-glucosidic mixed bonds and/or hemicellulose residues in molecules of polysaccharides the optical density-glucose concentration calibration function was preliminarily plotted with the aim to estimate the method line and quantitation limit. The plot linearity is maintained at the glucose content up to 1 mg/ml, then the plot achieves plateau. The calibration plot given in Figure 1 was used for the calculations.
[0077] At the first stage the study was carried out with 10% solution of (1→3)(1→4)(1→6)-β-D-glucans. This quantity of (1→3)(1→4)(1→6)-β-D-glucans imitates the minimum level of dishware soiling. α-amylase and β-1,3-1,4-glucanase were used as enzymes of the class of glucosyl hydrolases (Table 5).
Table 5 - Characteristics of glucosyl hydrolase from different sources
| No. | Glycosyl hydrolase enzyme | Source | Activity | pH range | Optimal temperature |
| 1 | α-Amylase | Microbiological (Bacillus licheniformis) | >100 U/g or >100 U/ml | 6.0-10.0 | 20-60°C |
| 2 | β-1,3-1,4-glucanase | Microbiological (Trichoderma reesei) | >140.000 U/g | 3.5-7.0 | 50-65°C |
| 3 | β-1,3-1,4-1,6-glucanase | Microbiological (Trichoderma reesei) | >20.000 U/g | 3.5-6.0 | 50-65°C |
| 4 | Licheninase | Microbiological (Bacillus spp.) | >100 U/g or >100 U/ml | 6.0-10.0 | 20-60°C |
| 5 | β-glucosidase | Microbiological (Aspergillus niger) | >4000 U/ml | 3.5-5.5 | 50-60°C |
[0078] 10% solution of β-glucans stabilized at 40 °C in water bath and adjusted to pH 7.0 was divided into 3 tubes: 1 - negative control (5 ml); 2 - with addition of 0.25 ml of α-amylase (10 ml); 3 - with addition of 0.75 ml of β-1,3-1,4-glucanase of the class of glucosyl hydrolases (30 ml), and thermostated during 30 minutes. At the end of this time pH in tubes No.2 and No.3 was adjusted to 4.0 with citric acid, then the tubes were centrifuged. The supernatant from tube No.3 was transferred to new tubes 3.1 and 3.2 by 10 ml into each with addition of 0.25 ml of deionized water and β-glucosidase, respectively. The tubes were thermostated during 30 minutes. Then 3 ml of the glucose oxidase reagent was added to all the tubes and after thermostating during 20 minutes optical density was measured at 510 nm wavelength. The reaction of the glucose oxidase reagent is shown in Figure 2. The composition of the glucose oxidase reagent is the following: glucose oxidase 15 000 IU/l, peroxidase 1 000 IU/l, phenol 11 mmol/l , 4-aminoantipyrine 0.77 mmol/l, chloric acid, Na2HPO4 buffer 150 mmol/l for pH 7.5 as well as glucose 5.55 mmol/l or 100 mg/dl for method calibration.
[0079] Glucose released from (1→3)(1→4)(1→6)-β-D-glucans was calculated by the calibrator or standard glucose solution according to the formula: glucose concentration (mg/dl) = (Asample/Acal) x standard concentration (mg/dl). The analytical factor for glucose was calculated according to the formula: glucose (mg/dl) x 0.05551 = Glucose (mmol/l). Thus, glucose quantity after enzymatic hydrolysis of (1→3)(1→4)(1→6)-β-D-glucans was determined and then it was recalculated and compared with activity of the enzyme of the class of glucosyl hydrolases.
[0080] According to the results of the conducted study it was established that the combination of enzymes of the class of glucosyl hydrolases, specifically α-amylase and β-1,3-1,4-glucanase, has synergistic activity and demonstrates glucose concentration increases by +15.1%, which speaks of degradation of beta-glucosidic bonds and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans. Individual enzymes α-amylase and β-1,3-1,4-glucanase show glucose concentration reduction and correspondingly degradation of glucans in connection with resorption of glucose residues by complex molecules of polysaccharides.
Table 6. Results of study of degradation of complex mixed glucans by means of enzymes
of the class of glucosyl hydrolases by UV spectrophotometry
| No. | Sample | Glucose concentration, mg/ml | |
| Absolute values | Relative values, % | ||
| 1 | Negative control (purified water) | 0.703 | - |
| 2 | α-Amylase | 0.567 | -19.3% |
| 3 | β-1,3-1,4-glucanase | 0.543 | -22.8% |
| 4 | α-Amylase + β-1,3-1,4-glucanase | 0.809 | +15.1% |
[0081] At the second stage the study was carried out with 70% solution of (1→3)(1→4)(1→6)-β-D-glucans from different sources. This quantity of (1→3)(1→4)(1→6)-β-D-glucans imitates a high level of soils on dishware for comprehensive estimation of activity of enzymes. High contents of (1→3)(1→4)(1→6)-β-D-glucans are fixed on dense surfaces in conditions of high water hardness of 10-42 dH, sorbing other soils back and reducing washing efficiency of the formulations, α-amylase, β-1,3-1,4-glucanase, β-1,3-1,4-1,6-glucanase, licheninase, β-glucosidase were used as enzymes of the class of glucosyl hydrolases (Table 6). 70% solution of β-glucans stabilized at 40 °C in water bath with pH adjusted to 7.0 was divided into 3 tubes: 1 - negative control (5 ml); 2 - with addition of 0.25 ml of α-amylase (10 ml); 3 - with addition of 0.750 ml of β-1,3-1,4-glucanase or 0.375 g of β-1,3-1,4-1,6-glucanase or 0.054 g of β-1,3-1,4-glucanase of the class of glucosyl hydrolases (30 ml), and thermostated during 30 minutes. At the end of this time pH of tubes No.2 and No. 3.1, 3.2, 3.3 was adjusted to 4.0 with citric acid, and then the tubes were centrifuged. The supernatant from tubes No.3.1, 3.2, 3.3 was transferred to new tubes by 10 ml to each with addition of 0.25 ml of deionized water, respectively. The tubes were thermostated during 30 minutes. Then 3 ml of the glucose oxidase reagent was added to all the tubes and after thermostating during 20 minutes optical density was measured at 510 nm wavelength. The reaction of the glucose oxidase reagent is shown in Figure 2.
[0082] According to the results of the second stage of the study it was established that the combination of enzymes of the class of glucosyl hydrolases, specifically α-amylase and β-1,3-1,4-glucanase or β-1,3-1,4-1,6-glucanase or licheninase or β-glucosidase, has synergistic activity and demonstrates significant glucose concentration increase, which speaks of effective degradation of beta-glucosidic bonds and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans. Individual enzymes of the class of glucosyl hydrolases show insignificant glucose concentration increase and correspondingly degradation of glucans in connection with resorption of glucose residues by complex molecules of polysaccharides, with the exception of highly active β-1,3-1,4-1,6-glucanase. Addition of α-amylase to glucosyl hydrolase, specifically to β-1,3-1,4-glucanase or licheninase or β-glucosidase, enables increasing degradation of (1→3)(1→4)(1→6)-β-D-glucans and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans, by +183.04%, +379.03% and +281.43%, respectively (Table 7). Addition of α-amylase to β-1,3-1,4-1,6-glucanase demonstrates increase in efficiency of degradation of (1→3)(1→4)(1→6)-β-D-glucans by +10.02% in connection with high efficiency of individual β-1,3-1,4-1,6-glucanase. A combination of two or more enzymes of the class of glucosyl hydrolases makes it possible to significantly increase efficiency of breakage of alpha- and beta-glucosidic bonds and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans of different origins.
Table 7. Result of the study of degradation of complex mixed glucans by means of enzymes
of the class of glucosyl hydrolases by UV spectrophotometry
| No. | Sample | Glucose concentration, mg/ml | |
| Absolute values | Relative values, % | ||
| 1 | Negative control (purified water) | 0.000±0.000 | - |
| 2 | α-Amylase | 0.015±0.010 | - |
| 3.1 | β-1,3-1,4-glucanase | 0.289±0.039 | +183.04% |
| 3.2 | β-1,3-1,4-glucanase + α-Amylase | 0.529±0.089 | |
| 4.1 | β-1,3-1,4-1,6-glucanase | 0.558±0.082 | +10.02% |
| 4.2 | β-1,3-1,4-1,6-glucanase + α-Amylase | 0.582±0.007 | |
| 5.1 | Licheninase | 0.124±0.028 | +379.03% |
| 5.2 | Licheninase + α-Amylase | 0.594±0.024 | |
| 6.1 | β-glucosidase | 0.140±0.001 | +281.43% |
| 6.2 | β-glucosidase + α-Amylase | 0.534±0.001 | |
[0083] The ability of combinations of enzymes to break alpha- and beta-glucosidic bonds and/or hemicellulose residues in molecules of mixed polysaccharides, specifically complex mixed glucans of cereal crops, starch, cell walls of plants and fungi, makes it possible to prevent resorption on smooth surfaces, remove tough fixed soils and stains on dense surfaces at lower temperatures in household detergents.
Example 5.
[0084] A study was conducted to determine efficiency of enzymes in the claimed composition as part of a dishwashing detergent for destruction of beta-1,3-glycosidic, beta-1,4-glycosidic, beta-1,6-glycosidic or alpha-1,4-glycosidic bonds in hemicellulose residues of polysaccharides consisting of glucose monomers or other monosaccharides that are contained in different parts of plants and cell walls of fungi and prevention of resorption after single dishwashing. The study was intended to test the hypothesis about improving removal of different plant-based soils during single dishwashing with the claimed combination of enzymes of the class of glucosyl hydrolases and alpha-amylase of the invention. The experimental detergent base was a gel-like universal dishwashing liquid specified in Table 2.
[0085] The analysis method is based on measuring the quantity of colored removed soils from dishware surface. 0.2 grams of cooked cereals enriched in beta-1,3- and beta-1,4-glycosidic bonds were evenly distributed with a brush on the inner side of the plate surface, leaving the edges clean. A wide brush was used to evenly distribute a thin layer and red food colorant E128 in order to mark out stains during preliminary dishwashing. Plates with soils were left to dry during 2-3 hours at room temperature for fixation of soils and imitation of home conditions. Then a wide container was taken, 2.5 liters of running water of 40°C, hardness 0-42 dH and a detergent with enzymes of the invention with concentration of 1 g/l of water were added therein and thoroughly mixed till complete detergent dissolution. The first container was filled with a dishwashing liquid without the composition of the invention only. A dishwashing liquid containing 0.0025% alpha-amylase was added to the second container. A dishwashing liquid containing 0.0025% alpha-amylase and 0.0025% beta-glucanase was added to the third container. Besides, efficiency of detergents with addition of cellulase was estimated. Then soiled plates were fully submerged in the containers and kept there for 1 minute. In 1 minute the plates were rinsed with water of room temperature, 1% alcoholic iodine solution was applied and distributed all over the inner side of the plate for better fixation of remaining polysaccharide soils with beta-1,3- and beta-1,4-glycosidic bonds. The iodine solution was washed off and the plates were again rinsed with water of room temperature. Similarly, the actions according to the method were repeated with lower enzyme concentrations and with their co-presence in a hand dishwashing liquid. Measurements were taken three times (n=3) for validity of the obtained data. The results were photographed for processing.
Results.
[0086] According to the results of estimating the increase of dishwashing detergent efficiency with respect to polysaccharide stains and soils based on rice, pasta, plant crops it was established that the claimed composition of the invention has high activity of degrading mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4- glucosidic bonds and/or hemicellulose residues. The components of the dishwashing detergent composition with alpha-amylase and licheninase enable achieving high efficiency with regard to improvement of hydrolysis of polysaccharides with mixed bonds and hemicellulose residues of shells of plant crops with the minimum concentration of composition components. A reduction in the quantity of dried cooked cereal remainders after single dishwashing was visually observed (Figure 3).
[0087] Single dishwashing with addition of the composition based on enzymes of the class of glucosyl hydrolases breaks a larger quantity of hemicellulose residues and mixed polysaccharides in grain fibers and scales, which partially consist of a sticky hydrocarbon known as starch. The combination of alpha-amylase and an enzyme of the class of glucosyl hydrolases has a synergistic effect and enables removal of soils without mechanical actions up to 90% when used in water with 0-42 dH hardness and at low temperatures and pH=7.5-8.5. Addition of 0.001% cellulase is beneficial in case of very dry cereal remainders.
Example 6.
[0088] A study was conducted to determine efficiency of the composition of the invention as part of tableted dishwasher powder after single use in quick cycles (15-30 minutes) and in conditions of low-temperature washing at 35-45°C. The study was intended to test the hypothesis about improvement of removal of different soils of superfood products (cereals, buckwheat, banana, mango, chia seeds, avocado pulp, amaranth, green peas) during single washing in an automatic dishwasher with the combination of enzymes of the class of glucosyl hydrolases of the invention. The experimental detergent was dry automatic dishwasher tablets specified in Table 3. The detailed chemical composition of selected superfood soils is given in Table 8 as the detergent is intended for a wide range of soils and stains.
Table 8. Chemical composition of superfood soils with specification of basic chemical
bonds in prevailing molecules of hydrocarbons
| Superfood product | Basic chemical substances | Basic chemical bonds in hydrocarbons |
| Cereals | Starch: 50-70 % | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Proteins:7-15 % | ||
| Monosaccharides: 2.6-13.6% | ||
| Fiber: 2.1-14.0 % | ||
| Oligosaccharides: 2.0-5 % | ||
| Lipids: 2.0-5.5 % | ||
| Ash: 1.3-6.2% | ||
| Buckwheat | Starch: 61-62% | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Fiber: 12-15% | ||
| Proteins: 8-16 % | ||
| Fructose: 3.1 % | ||
| Glucose: 3 % | ||
| Mono- and disaccharides: 3 % | ||
| Fats: 2.5-2.9 % | ||
| Banana | Glucose: 49.8 % | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Mono- and disaccharides: 38 % | ||
| Fructose: 14 % | ||
| Starch: 1.2% | ||
| Ash: 0.8 % | ||
| Fiber: 0.6 % | ||
| Fats: 0.6 % | ||
| Mango | Mono- and disaccharides: 27.3 % | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Fiber: 8 % | ||
| Proteins: 1.5-5.5 % | ||
| Fats: 0.7 % | ||
| Chia seeds | Fats: 30-33 % | β-1,4- glycosidic bonds |
| Fiber: 18-30 % | ||
| Proteins: 15-25 % | ||
| Phenolic compounds: 8.8-9 % | ||
| Avocado | Fiber: 20% | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Fats: 15.3% | ||
| Mono- and disaccharides: 10% | ||
| Purines: 10% | ||
| Proteins: 2.8% | ||
| Glucose: 2.5 % | ||
| Fructose: 0.5 % | ||
| Amaranth seeds | Starch: 60 % | β-1,4-, α-1,2- glycosidic bonds |
| Proteins: 14-20 % | ||
| Fats: 10 % | ||
| Sucrose: 3 % | ||
| Green peas | Purine bases: 56 % | α-1,4-, α-1,6-, β-1,4-, β-1,3-, β-1,6- glycosidic bonds |
| Proteins: 30.7 % | ||
| Glucose: 21 % | ||
| Mono- and disaccharides: 12.6% | ||
| Pectin: 12 % | ||
| Fructose: 7.1 % | ||
| Fats: 1.2% |
[0089] There were comparative tests of 4 experimental samples of dishwasher tablets to estimate dishwashing efficiency based on 8 kinds of soil combinations and resorption of soils on clean dishes after cycle completion. To estimate absence of soil transfer to clean dishware 2 kinds of clean dishware (mugs, plates) were put into a dishwasher and mixed with soiled dishware. Then, upon washing cycle completion, presence of streaks, drops, water stains or film on the initially clean dishware was estimated. Presence of slightest spots or stains and absence of shine spoke of formation of possible deposits or unsatisfactory efficiency of the detergent. At the end of study the best composition of enzymes within the composition of the invention that had the maximum range of actions on different soils enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4- glucosidic bonds and/or hemicellulose residues was selected.
[0090] The washing results were estimated by visualization on a 5-score scale in comparison with the initial data:
5 - no remaining soils
4 - soil traces remaining
3 - 10-60% soils remaining
2 - 70-80% soils remaining
1 - 85-90% soils remaining
0 - all soils remained unchanged
Results.
[0091] According to the results of estimating efficiency of removal of superfood product soils, giving a shine to dishware it was established that the claimed composition based on enzymes of the class of glucosyl hydrolases in combination with alpha-amylase as part of dishwasher tablets has high efficiency in removal of dry soils (oatmeal, buckwheat, amaranth, chia seeds), tough protein soils (green peas puree) as well as medium efficiency in removal of oily soils (avocado, banana). After 1 wash cycle no streaks, stains, drops, limescale remained on dishware. A reduction in the quantity of tough, burnt-in or oily soils with the use of the combination of alpha-amylase and a supplemental enzyme of the class of glucosyl hydrolases was visually observed (Figure 4).
[0092] The claimed composition with addition of enzymes of the class of glucosyl hydrolases in combination with alpha-amylase as part of automatic dishwasher tablets has high efficiency with respect to superfood soils enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4- glucosidic bonds and/or hemicellulose residues in conditions of low-temperature dishwashing (40-45°C), in hard water conditions (0-42 dH) and in short washing cycles (15-30 minutes) at pH=10.5-12.5. A reduction in the quantity of tough (burnt-in) soils of superfood products was visually observed (Figure 5).
[0093] The claimed composition with addition of 0.002% beta-glucanase and 0.002% alpha-amylase as part of automatic dishwasher tablets leaves the least quantity of deposits and soils on dishware, providing for 85-90% efficiency of removing soils. Addition of 0.002% cellulase in dishwasher tablets removes 10-15% of superfood soils on dishware, while the base of universal automatic dishwasher tablets demonstrates the worst results of removing tough (burnt-in) soils and leaves 30-40% of soils (Table 9).
Table 9. Efficiency of removal of 8 kinds of soil combinations
| Product | Soil combinations | Visual estimation | |||
| By each soil | Total of 5 scores | Total | Effect, % | ||
| Cereals | |||||
| Product base | Oatmeal | 2.0 | 2.0 | 4.0 of 10 | 40.0% |
| Buckwheat | 2.0 | ||||
| Product base + 0.002% amylase | Oatmeal | 3.0 | 3.5 | 5.5 of 10 | 55.0% |
| Buckwheat | 2.5 | ||||
| Product base + 0.002% amylase + 0.002% beta-glucanase | Oatmeal | 5.0 | 4.75* | 9.5 of 10 | 95.0% |
| Buckwheat | 4.5 | ||||
| Superfood soils | |||||
| Product base | Avocado | 2.0 | 2.8 | 17 of 30 | 57.0% |
| Mango | 3.0 | ||||
| Banana | 2.0 | ||||
| Chia | 4.0 | ||||
| Amaranth | 3.0 | ||||
| Green peas puree | 3.0 | ||||
| Product base + 0.002% alpha-amylase | Avocado | 2.0 | 3.0 | 22 of 30 | 73.0% |
| Mango | 4.0 | ||||
| Banana | 2.0 | ||||
| Chia | 5.0 | ||||
| Amaranth | 4.0 | ||||
| Green peas puree | 5.0 | ||||
| Odorless dishwashing liquid base + 0.002% beta-glucanase + 0.002% alpha-amylase | Avocado | 3.5 | 4.5* | 27 of 30 | 90.0% |
| Mango | 5.0 | ||||
| Banana | 3.5 | ||||
| Chia | 5.0 | ||||
| Amaranth | 5.0 | ||||
| Green peas puree | 5.0 | ||||
| Odorless dishwashing liquid base + 0.002% cellulase + 0.002% alpha-amylase | Avocado | 2.5 | 4.0* | 24 of 30 | 80.0% |
| Mango | 5.0 | ||||
| Banana | 2.5 | ||||
| Chia | 5.0 | ||||
| Amaranth | 5.0 | ||||
| Green peas puree | 4.0 | ||||
| *a statistically significant result (p<0.05) | |||||
[0094] The composition of the invention is efficient in prevention of transfer of washed soils to clean dishware during washing, i.e. it has anti-resorption properties. The base of dishwasher tablets has rather low efficiency and contributes to resorption of soils based on mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4-glucosidic bonds and/or hemicellulose residues on the rear side of clean dishware.
Example 7.
[0095] A study was conducted to determine efficiency of the composition of the invention as part of tableted dishwasher powder after single use in a quick wash cycle of 30 minutes and in conditions of low-temperature washing at 45°C and pH=10.5-12.5. The study was intended to test the hypothesis about anti-resorbing properties and absence of transfer of soils to clean dishware after removal of different soils from products during single automatic dishwashing with the combination of enzymes of the class of glucosyl hydrolases of the invention. The experimental detergent was dry automatic dishwashing tablets specified in Table 3.
[0096] There were comparative tests of experimental samples of dishwasher tablets to estimate resorption of soils on clean dishware at the cycle end. Running water hardness was 13-15dH. 4 kinds of soils were used (concentrated black tea, a cooked mixture of cereals, baked egg yolk, burnt-in vegetable oil of 82% fat content) on 4 kinds of dishware (tea cups, glazed earthenware plates, glass plates and stainless steel plates). In addition, ballast soil (~50% fat content) in the quantity of 50 g per cycle, namely a mixture of sunflower oil, fats, eggs, cream, milk, mayonnaise, flour and mashed potatoes, was used. In each cycle 3 soiled pieces of each kind were used. Then, upon washing cycle completion, presence of streaks, drops, water stains or cloudy film on the initially clean dishware was estimated. Presence of slightest dark spots or stains, absence of shine spoke of formation of possible deposits or unsatisfactory efficiency of the detergent. Upon study completion the best combination of enzymes within the composition of the invention that had maximum anti-resorbing action after removal of different soils enriched in mixed homo- and heteropolysaccharides with α-1,4-, α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues was selected.
[0097] To estimate absence of transfer of soils to clean dishware 4 kinds of clean dishware similar to the soiled one were put into a dishwasher and mixed with soiled dishware. The results of anti-resorbing action were estimated on a 5-score scale in comparison with the initial data:
5 - soils were not resorbed;
4 - 5-10% soils were resorbed;
3 - 10-60% soils were resorbed;
2 - 70-80% soils were resorbed;
1 - 85-90% soils were resorbed;
0 - all soils were resorbed.
Results.
[0098] According to the results of estimation of anti-resorption action, prevention of redeposition of soils enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4- glucosidic bonds and/or hemicellulose residues and giving a shine to dishware it was established that the experimental composition based on enzymes of the class of glucosyl hydrolases in combination with alpha-amylase as part of dishwasher tablets has high efficiency of protection against resorption of removed soils. The composition based on glucosyl hydrolase and alpha-amylase of the invention acts as an inhibitor of redeposition of soils. After 1 cycle of washing with a detergent containing the composition of two enzymes of the invention no streaks, stains, drops, limescale remain on dishware and there is no resorption of soils on clean dishware, glass dishware remains transparent. A reduction in the quantity of tough, burnt-in and oily soils with the use of the combination of alpha-amylase and a supplemental enzyme of the class of glucosyl hydrolases was visually observed (Table 10).
Table 10. Estimation of transfer of soils to clean dishware by 4 kinds of dishware
| Composition components | Dishware | Visual estimation on a 5-score scale | ||
| By each soil | Total of 5 | Total of 20 | ||
| Tableted detergent No. 1 | ||||
| No | Cup | 5.0 | 4.4 | 17.7 |
| Earthenware plate | 5.0 | |||
| Glass plate | 4.0 | |||
| Stainless steel plate | 3.7 | |||
| Tableted detergent No.2 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 4.6 | 18.3 |
| Earthenware plate | 5.0 | |||
| Glass plate | 4.3 | |||
| Stainless steel plate | 4.0 | |||
| Tableted detergent No.3 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 5.0 | 20.0 |
| Earthenware plate | 5.0 | |||
| Beta-glucanase 0.05% | Glass plate | 5.0 | ||
| Stainless steel plate | 5.0 | |||
| Tableted detergent No.4 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 5.0 | 20.0 |
| Earthenware plate | 5.0 | |||
| Cellulase 0.05% | Glass plate | 5.0 | ||
| Stainless steel plate | 5.0 | |||
[0099] The experimental composition with addition of enzymes of the class of glucosyl hydrolases in combination with alpha-amylase as part of automatic dishwasher tablets does not leave streaks after removal of soils enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3- β-1,4- glucosidic bonds and/or hemicellulose residues in conditions of low-temperature wash cycles (45°C), in hard water and in short wash cycles (30 minutes) at pH=10.5-12.5. A reduction of streaks was visually observed, with the increase of the average score from 3.4 to 5.0 and total score from 17.0 to 20.0 (Table 11).
Table 11. Estimation of presence/absence of streaks on 4 kinds of clean dishware
| Composition components | Dishware | Visual estimation on a 5-score scale | ||
| By each soil | Total of 5 | Total of 20 | ||
| Tableted detergent No. 1 | ||||
| No | Cup | 5.0 | 3.4 | 17.0 |
| Earthenware plate | 5.0 | |||
| Glass plate | 4.0 | |||
| Stainless steel plate | 3.0 | |||
| Tableted detergent No.2 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 3.5 | 17.7 |
| Earthenware plate | 5.0 | |||
| Glass plate | 4.7 | |||
| Stainless steel plate | 3.3 | |||
| Tableted detergent No.3 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 5.0 | 20.0 |
| Earthenware plate | 5.0 | |||
| Beta-glucanase 0.05% | Glass plate | 5.0 | ||
| Stainless steel plate | 5.0 | |||
| Tableted detergent No.4 | ||||
| Alpha-amylase 0.05% | Cup | 5.0 | 5.0 | 20.0 |
| Earthenware plate | 5.0 | |||
| Cellulase 0.05% | Glass plate | 5.0 | ||
| Stainless steel plate | 5.0 | |||
Example 8.
[0100] There was a laboratory study of washing and stain-removing efficiency of components of the composition of the invention as part of a concentrated liquid laundry detergent for white and colored fabrics specified in Table 12 with respect to stains enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues in conditions of low-temperature wash cycles at pH=8.0-9.5.
Table 12. Ingredients of a concentrated gel laundry detergent for white and colored
fabrics with the claimed composition of the invention
| No. | Component | Content, % wt. |
| 1 | Purified water | up to 100.00 |
| 2 | Alkyl polyethylene glycol sulfate with the general formula R1-O(-CH2-CH2-O)n1 (SO3) n2 X1, where n1 takes values from 0 to 10 and denotes the number of polyethylene groups, R1 represents an alkyl and/or alkenyl group with a hydrocarbon chain length from 5 to 22 carbon atoms, n2 takes values from 0 to 1 and denotes the number of sulfate groups , X1 represents a cation of an alkali and/or alkali-earth metal, ammonium, alkylammonium, alkanolammonium, glucoammonium, basic aminoacid | 2.5-10.00 |
| 3 | Alkylglucoside with the general formula: R28-O-[G]p3, where R28 is an alkyl and/or alkenyl group with a hydrocarbon chain length from 4 to 22 carbon atoms, G is a saccharide fragment comprising 5 or 6 carbon atoms, p3 can take values from 1 to 4, specifically C10-16 alkylpolyglucoside and C8-10 alkylpolyglucoside | 1.5-10.00 |
| 4 | Salts of higher carboxylic acids with the general formula: R1-CO2X1, where R1 is an alkyl and/or alkenyl group with a hydrocarbon chain length from 5 to 21 carbon atoms, and X1 is a cation of an alkali and/or alkali-earth metal, ammonium, alkylammonium, alkanolammonium, glucoammonium, basic aminoacid; | 1.0-5.00 |
| 5 | Glucosyl hydrolase, for example, beta-glucanase - expressed as active substance | 0.001-0.1 |
| 6 | Alpha-amylase - expressed as active substance | 0.0025-0.1 |
| 7 | Complexing agent MGDA | 0.1-1.50 |
| 8 | Glycerin of natural origin | 1.0-5.00 |
| 9 | Sodium chloride | 0.05-5.00 |
| 10 | Cotton seed extract | 0.005-0.50 |
| 11 | Preservative | 0.005-0.75 |
| 12 | Sodium hydroxide or potassium hydroxide for pH regulation | 0.005-1.00 |
| 13 | pH regulator, for example, citric acid or another acid | 0.005-1.00 |
| 14 | Auxiliary substances, if required | 0.10-10.00 |
[0101] The test methods were based on recognized recommendations of the European Association A.I.S.E. [A.I.S.E. Laundry Detergent Testing Guidelines Minimum requirements for comparative detergents testing https://www.aise.eu/documents/document/20180625164030-laundry_detergent_testing_guidelines_v_5_2_june_2018_.pdf] for determination of washing efficiency with account for the degree of removal of stains (SRI) and L,a,b values. SRI index was calculated according to the formula from ASTM D 4265 Standard guide for evaluating stain removal performance in home laundering [DOI: 10.1520/D4265-14]. The guide was prepared by Committee D-12 for soaps and other detergents of the American Society for Testing and Materials (ASTM) and published in 2014. This test enables estimation of efficiency of the composition by the degree of removal of fresh and old stains of different origin from dense surfaces, specifically from cotton and synthetic materials imitating actual laundering conditions. Samples were washed in a Linitest laboratory washing machine. The test conditions were widely spread recommendation in the EU territory, specifically temperature 40°C, water hardness 11.2° dH and the standard wash cycle (mixed fabrics). The detergent consumption was 10 g/l.
[0102] The test was carried out for tough bio-stains of different origin enriched in mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues (Table 13). The selected tough bio-stains enabled estimation of both general washing efficiency of the formulation and efficiency with respect to individual soils due to presence of glucosyl hydrolase and alpha-amylase of the invention.
Table 13. Tested bio-stains and bio-soils from A.I.S.E.
| Name | Content | Type |
| KC-H087 | Cooked cereals with chocolate | Enzymatic* |
| *contains mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues. | ||
[0103] SRI is the quantitative index of removal of soils and stains in conventional units. The values are based on parameters of whiteness L and color a,b, are calculated mathematically according to the special formula from the standard ASTM D 4265-14 [DOI: 10.1520/D4265-14.]:
where: L is reflectance, a - redness/greenness ratio, b - yellowness/blueness ratio, c - unstained fabric washed in laundering conditions, w - washed stained fabric.
[0104] The method has many advantages, specifically high accuracy, reproducibility, a low error (below 5%), it takes into account color of fabrics and its change during washing, different types of fabrics and is closest to actual visual perception; it also levels out action of optical dyes widely spread in compositions of laundry detergents. An effective and statistically significant difference is the difference of 2 and more SRI units. According to practical experience, 1 SRI unit corresponds to 5% of additional washing efficiency and contributes to the general efficiency of the formulation.
Results.
[0105] According to the results of efficiency and speed of removal of tough stains based on mixed homo- and heteropolysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues it was established that the combination of an enzyme of the class of glucosyl hydrolases and alpha-amylase as part of a concentrated laundry detergent for white and color fabrics has a well-expressed stain removal performance with respect to tough soils comparing to the reference detergent that does not contain glucosyl hydrolase and alpha-amylase in the combination of the invention.
[0106] At the end of study there were obvious changes of the estimated index of performance of removal of tough stains based on different organic compounds, including polysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues. According to the index dynamics, addition of highly-efficient alpha-amylase and glucosyl hydrolase in co-presence increases the degree of removal of tough stains of polysaccharide origin. Addition of 0.05% beta-glucanase of the class of glucosyl hydrolases in combination with 0.05% alpha-amylase made it possible to increase the SRI index by 5.2 units (+6.45%) comparing to the base of a laundry gel for white and color fabrics without the composition of the invention, which exceeds the recommended limit ΔSRI>2. A comparable result was obtained with addition of 0.05% cellulase of the class of glucosyl hydrolases in combination with 0.05% alpha-amylase, which enabled increasing the SRI index by 4.7 units (+5.83%) comparing to the base of a laundry gel for white and color fabrics without the composition of the invention, which exceeds the recommended limit ΔSRI>2. Alpha-amylase in combination with glucosyl hydrolase made it possible to significantly increase stain removal performance as part of a universal liquid laundry detergent for white and color fabrics. It is interesting that addition of alpha-amylase only reduced removal of stains based on polysaccharides with α-1,4- and α-1,6-, β-1,3-, β-1,4-glucosidic bonds and/or hemicellulose residues, worsening stain removal performance by 6.7 SRI units statistically significant, p<0.05). Individual enzymes of the class of glucosyl hydrolases also did not demonstrate high stain removal performance visible to the human eye. The sample of the detergent base without the composition of the invention did not show very high performance of removal of selected stains and soils, which speaks of insufficient efficiency of surfactants in removal of stains and soils in the laundering process and the need in additional enzymes with targeted action (Table 14).
Table 14. Estimation of stain and soil removal performance
| Tested sample | Active components | KC-H087 | ||
| SRI, absolute value | Change, % | |||
| Initial soil | 53.2 | - | - | |
| Sample of a laundry gel for white and color fabrics No.1 | - | 80.6 | - | - |
| Sample of a laundry gel for white and color fabrics No.2 | α-Amylase 0.05% | 73.9 | -6.7 | -8.31% |
| Sample of a laundry gel for white and color fabrics No.3 | Licheninase 0.05% | 82.3 | +1.7 | +2.11% |
| Sample of a laundry gel for white and color fabrics No.4 | Cellulase 0.05% | 80.4 | -0.2 | -0.25% |
| Sample of a laundry gel for white and color fabrics No.5 | β-glucosidase 0.05% | 71.4 | -9.2 | -11.41% |
| Sample of a laundry gel for white and color fabrics No.6 | β-glucanase 0.05% | 79.8 | -0.8 | -1.00% |
| Sample of a laundry gel for white and color fabrics No.7 | α-Amylase 0.05% | 85.8 ** | +5.2 | +6.45% |
| β-glucanase 0.05% | ||||
| Sample of a laundry gel for white and color fabrics No.8 | α-Amylase 0.05% | 85.3** | +4.7 | +5.83% |
| Cellulase 0.05% | ||||
| *mean significant difference, statistically significant (p<0.05), increase of standard
washing efficiency by more than 5%; **mean significant difference, statistically significant
(p<0.05), increase of standard washing efficiency by more than 10%; **best significant difference, statistically significant (p<0.05), increase of standard washing efficiency by more than 20% |
Example 9.
[0107] There was a laboratory study of stability of enzymes of the composition of the invention, specifically alpha-amylase and glucosyl hydrolase, in a concentrated foam dishwashing liquid specified in Table 1.
[0108] The test was carried out in a laboratory according to the company's own methods for determination of activity of enzymes after 4 weeks in a chamber at 30° C. The test makes it possible to estimate maintaining of activity of enzymes in the base of a ready product according to the method of accelerated stability during thermostating to reveal residual activity throughout the shelf life and, correspondingly, product performance. Samples of a concentrated foam dishwashing liquid with pH 10.0 specified in Table 1 were prepared and alpha-amylase and glucosyl hydrolase separately and in co-presence were added. The samples were thermostated in an oven during 4 weeks, and then activity of enzymes was estimated according to developed methods for each enzyme. The test makes it possible to reveal any changes in activity of sensitive enzymes from the group of glucosyl hydrolases, including alpha-amylase. The residual activity estimation error is ±5%. The tests were carried out once (n=1). In addition, activity of enzymes at different pH, specifically at pH 6.0, 8.0 and 10.0, after 4 weeks was estimated to model behavior of enzymes and stability in different household detergents.
Results.
[0109] According to the results of estimation of residual activity of enzymes it was established that the tested alpha-amylase, different glucosyl hydrolases and the combination of enzymes maintain their activity in conditions of the accelerated stability method (Table 13). The tested composition based on alpha-amylase and exo or endo glucosyl hydrolase as part of a concentrated foam dishwashing liquid maintains its high activity after 2 and 4 weeks of accelerated storage, which speaks of the possibility of co-presence of the two enzymes in formulations of household detergents and maintaining of high performance values demonstrated in Examples 5-8. The composition based on alpha-amylase and different glucosyl hydrolases displays its activity in systems with basic pH above 8.0, namely 10.0 and above, while activity of many enzymes decreases in an acid pH area below 6.0, which limits the field of use (Table 15). The authors surprisingly discovered that the composition based on glucosyl hydrolases maintains its activity within the pH range of 10 and above, which is optimal for most dishwasher, hand dishwashing and laundry detergents.
Table 15. Residual activity of composition enzymes after accelerated storage
| Experimental sample | Composition components | Residual activity of enzymes at pH 10, % | ||
| 0 week | 2 weeks | 4 weeks | ||
| Activity of individual enzymes | ||||
| Sample No.1 | Alpha-amylase 0.05% | 100% | 96% | 95% |
| Sample No.2 | Laminarinase 0.05% | 100% | 93% | 92% |
| Sample No.3 | Cellulase 0.05% | 100% | 100% | 100% |
| Sample No.4 | β-glucosidase 0.05% | 100% | 97% | 97% |
| Sample No.5 | β-1,3-1,4-glucanase 0.05% | 100% | 97% | 95% |
| Sample No.6 | β-1,3-1,4-1,6-glucanase 0.05% | 100% | 98% | 95% |
| Activity of alpha-amylase in presence of glucosyl hydrolase | ||||
| Sample No.7 | α-Amylase 0.05% | 100% | 94% | 93% |
| β-1,3-1,4-glucanase 0.05% | ||||
| Sample No.8 | α-Amylase 0.05% | 100% | 94% | 93% |
| β-1,3-1,4-1,6-glucanase 0.05% | ||||
| Sample No.9 | α-Amylase 0.05% | 100% | 92% | 92% |
| Laminarinase 0.05% | ||||
| Sample No.10 | α-Amylase 0.05% | 100% | 95% | 92% |
| Cellulase 0.05% | ||||
| Sample No.11 | α-Amylase 0.05% | 100% | 93% | 91% |
| β-glucosidase 0.05% | ||||
Table 16. Residual activity of composition enzymes at different pH
| Experimental sample | Composition components | Residual activity of enzymes, % | ||
| pH 6.0 | pH 8.0 | pH 10.0 | ||
| Activity of individual enzymes | ||||
| Sample No.1 | Alpha-amylase 0.05% | 72% | 85% | 95% |
| Sample No.2 | Laminarinase 0.05% | 60% | 82% | 92% |
| Sample No.3 | Cellulase 0.05% | 58% | 81% | 100% |
| Sample No.4 | β-glucosidase 0.05% | 60% | 77% | 97% |
| Sample No.5 | β-1,3-1,4-glucanase 0.05% | 69% | 75% | 95% |
| Sample No.6 | β-1,3-1,4-1,6-glucanase 0.05% | 50% | 73% | 95% |
| Activity of alpha-amylase in presence of glucosyl hydrolase | ||||
| Sample No.7 | α-Amylase 0.05% | 55% | 78% | 93% |
| β-1,3-1,4-glucanase 0.05% | ||||
| Sample No.8 | α-Amylase 0.05% | 47% | 71% | 93% |
| β-1,3-1,4-1,6-glucanase 0.05% | ||||
| Sample No.9 | α-Amylase 0.05% | 52% | 78% | 92% |
| Laminarinase 0.05% | ||||
| Sample No.10 | α-Amylase 0.05% | 51% | 80% | 92% |
| Cellulase 0.05% | ||||
| Sample No.11 | α-Amylase 0.05% | 53% | 73% | 91% |
| β-glucosidase 0.05% | ||||
[0110] The composition of the concentrated foamy dishwashing liquid based on alpha-amylase and glucosyl hydrolase maintains its activity during the product shelf life for achievement of high results for breakage of alpha- and beta-glycosidic mixed bonds and/or hemicellulose residues in molecules of polysaccharides for prevention of resorption on smooth surfaces. Alpha-amylase is stable in presence of glucosyl hydrolases thanks to the selected combinations of formulations given in Examples 1-4 and selection of special system pH. Since alpha-amylase is very sensitive, demonstrates low activity at pH above 8.0 and instability at high temperatures from 30 to 60°C, the results provided above speak of a new technical result achieved by raw material characteristics and the possibility of co-use for achievement of claimed effects of the composition of the invention.
1. A cleaning composition comprising the following glycosyl hydrolases:
(A) a glycosyl hydrolase targeting (1,4)-alpha-D-glucoside bonds, preferably alpha-amylase; and
(B) and at least one glycosyl hydrolase targeting beta-glycoside bonds selected from beta-glucanase, beta-glucosidase, cellulase, licheninase or laminarinase; and
wherein the mass ratio of (A) and (B) in said cleaning composition is (0.0025-0.25):(0.0010-0.25), respectively.2. The cleaning composition of claim 1, wherein said at least one glycosyl hydrolase
targeting beta-glycoside bonds is selected from the following or a combination thereof:
beta-1,3-1,4-glucanase, beta-1,3-1,4-1,6-glucanase, licheninase, beta-glucosidase, cellulase.
beta-1,3-1,4-glucanase, beta-1,3-1,4-1,6-glucanase, licheninase, beta-glucosidase, cellulase.
3. The cleaning composition of claim 1, wherein the pH of said composition is in the
range of 9.5-13, preferably 10.0-12.5.
4. The cleaning composition of any of the preceding claims, wherein said cleaning composition
is a liquid or gel cleaning dishwashing composition and preferably comprises 0.0025-0.05
% wt. of (A) and 0.001-0.10 % wt. of (B).
5. The liquid or gel cleaning composition of claim 4 for use in an aqueous dishwashing
solution, preferably for use in an aqueous manual hand dishwashing solution, for dishwashing
- at water hardness less than 42 dH, preferably 13-15dH,
- at any dishwashing temperature from +10°C to +45°C, and
- at pH=6.5-9.5, preferably at pH=7.5-8.5.
6. Use of the liquid or gel cleaning dishwashing composition of claim 4 as per an aqueous
dishwashing solution, preferably as per an aqueous manual hand dishwashing solution,
for dishwashing
- at water hardness less than 42 dH, preferably 13-15dH,
- at any dishwashing temperature from +10°C to +45°C, and
- at pH=6.5-9.5, preferably at pH=7.5-8.5.
7. The cleaning composition of any of claims 1-3, wherein said cleaning composition is
in powder, granulate, capsule or tablet form and preferably comprises 0.0025-0.05
% wt. of (A) and 0.001-0.10 % wt. of (B).
8. The cleaning composition of claim 7 for use in an aqueous dishwashing solution, preferably
for use in an automatic or non-hand dishwashing solution, for dishwashing
- at water hardness less than 42 dH, preferably 13-15dH,
- at any dishwashing temperature from +40°C to +65°C, +40°C to +45°C, or +35°C to +45°C, and
- at pH=9.5-13.5, preferably at pH=10.5-12.5.
9. Use of the cleaning composition in powder, granulate, capsule or tablet form of claim
7 as per an aqueous dishwashing solution, preferably as an automatic or non-hand dishwashing
solution, for dishwashing
- at water hardness less than 42 dH, preferably 13-15dH,
- at any dishwashing temperature from +40°C to +65°C, +40°C to +45°C, or +35°C to +45°C, and
- at pH=9.5-13.5, preferably at pH=10.5-12.5.
10. The cleaning composition of any of claim 1-3, wherein said cleaning composition is
in an aqueous spray solution form and preferably comprises 0.0025-0.05 % wt. of (A)
and 0.001-0.10 % wt. of (B).
11. The cleaning composition of claim 10 for use in or as a surface cleaner, preferably
for use in or as a hard surface cleaner, further preferably for use in or as a kitchen
surface cleaner, for cleaning said surfaces
- at any application temperature from +10°C to +35°C, and
- at pH=6-9, preferably at pH=7-8.
12. Use of the cleaning composition of claim 10 as per a surface cleaner, preferably as
a hard surface cleaner, further preferably as a kitchen surface cleaner, for cleaning
said surfaces
- at any application temperature from +10°C to +35°C, and
- at pH=6-9, preferably at pH=7-8.
13. The cleaning composition of any of claim 1-3, wherein said cleaning composition is
in liquid, gel, powder, granulate, capsule or tablet form and preferably comprises
0.0025-0.1 % wt. of (A) and 0.001-0.10 % wt. of (B).
14. The cleaning composition of claim 13 for use in an aqueous laundry composition for
laundry washing
- at water hardness less than 42 dH, preferably 11-15dH,
- at any laundry washing temperature from +40°C to +65°C, +40°C to +45°C, or +35°C to +45°C, and
- at pH=7-11, preferably at pH=8.0-9.5.
15. Use of the cleaning composition of claim 13 in an aqueous laundry composition for
laundry washing
- at water hardness less than 42 dH, preferably 11-15dH,
- at any laundry washing temperature from +40°C to +65°C, +40°C to +45°C, or +35°C to +45°C, and
- at pH=7-11, preferably at pH=8.0-9.5.
REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
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