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Title:
METHOD OF PRODUCING ANTIMICROBIAL EXTRACT FROM CHICORY
Document Type and Number:
WIPO Patent Application WO/2023/170340
Kind Code:
A1
Abstract:
The present disclosure relates to a method for producing an antimicrobial extract from chicory biomass. The disclosure further concerns a chicory extract having antimicrobial activity obtainable with said method. The present disclosure further concerns products comprising the obtained chicory extract having antimicrobial activity. The present disclosure concerns the use of said chicory extract in cosmetic products, hygiene products, personal care products or health care products.

Inventors:
HÄKKINEN SUVI (FI)
NOHYNEK LIISA (FI)
SIIKA-AHO MATTI (FI)
OKSMAN-CALDENTEY KIRSI-MARJA (FI)
Application Number:
PCT/FI2023/050132
Publication Date:
September 14, 2023
Filing Date:
March 09, 2023
Export Citation:
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Assignee:
TEKNOLOGIAN TUTKIMUSKESKUS VTT OY (FI)
International Classes:
A01N65/12; A01N25/02; A01P1/00; A61K8/9789; A61K36/28
Foreign References:
US4886672A1989-12-12
CN110156918A2019-08-23
CN108244658A2018-07-06
CN107349124A2017-11-17
Other References:
EDITH LECLERCQ: "Sesquiterpene Lactones and Inulin from Chicory Roots: Extraction, Identification, Enzymatic Release and Sensory Analysis", 1 January 1992 (1992-01-01), XP055316456, Retrieved from the Internet [retrieved on 20161103]
JASKIEWICZ ANDRZEJ ET AL: "Novel Biodegradable Starch Film for Food Packaging with Antimicrobial Chicory Root Extract and Phytic Acid as a Cross-Linking Agent", FOODS, vol. 9, no. 11, 19 November 2020 (2020-11-19), pages 1696, XP093046960, DOI: 10.3390/foods9111696
MILICA BOGDANOVIC, KATARINA CANKAR, SLADANA TODOROVIC, MILAN DRAGICEVIC, ANA SIMONOVIC, ADELE VAN HOUWELINGEN, ELIO SCHIJLEN, BERT: "Tissue specific expression and genomic organization of bitter sesquiterpene lactone biosynthesis in Cichorium intybus L", INDUSTRIAL CROPS AND PRODUCTS, vol. 129, 2019, pages 253 - 260, XP085578162, ISSN: 0926-6690, Retrieved from the Internet DOI: 10.1016/j.indcrop.2018.12.011
CANAVEZ, A. D. P. M.DE OLIVEIRA PRADO CORREA, G.ISAAC, V. L. B.SCHUCK, D. C.LORENCINI, M.: "Integrated approaches to testing and assessment as a tool for the hazard assessment and risk characterization of cosmetic preservatives", JOURNAL OF APPLIED TOXICOLOGY, vol. 41, no. 10, 2021, pages 1687 - 1699, Retrieved from the Internet
CANKAR, K.BUNDOCK, P.SEVENIER, R.HAKKINEN, S.T.HAKKERT, J.H.BEEKWILDER, J.VAN DER MEER, I.M.DE BOTH, M.BOSCH, D.: "Inactivation of the germacrene A synthase genes by CRISPR/Cas9 eliminates the biosynthesis of sesquiterpene lactones in Cichorium intybus L", PLANT BIOTECHNOL. J., 2021, Retrieved from the Internet
HALLA, N.FERNANDES, I.P.HELENO, S.A.COSTA, P.BOUCHERIT-OTMANI, Z.BOUCHERIT, K.RODRIGUES, A.E.FERREIRA, I.C.F.R.BARREIRO, M.F.: "Cosmetics Preservation: A Review on Present Strategies", MOLECULES, vol. 23, 2018, pages 1571, XP055588453, Retrieved from the Internet DOI: 10.3390/molecules23071571
HOUSHYANI, B., KABOUW, P., MUTH, D.: "Characterization of the natural variation in Arabidopsis thaliana metabolome by the analysis of metabolic distance", METABOLOMICS, vol. 8, 2012, pages 131 - 145, Retrieved from the Internet
ARJEN LOMMEN: "MetAlign: Interface-Driven, Versatile Metabolomics Tool for Hyphenated Full-Scan Mass Spectrometry Data Preprocessing", ANALYTICAL CHEMISTRY, vol. 81, no. 8, 2009, pages 3079 - 3086, XP055050688, DOI: 10.1021/ac900036d
LIISA J. NOHYNEKHANNA-LEENA ALAKOMIMARJA P KÄHKÖNENMARINA HEINONENILKKA M. HELANDERKIRSI-MARJA OKSMAN-CALDENTEYPIITTA H. PUUPPONEN: "Berry Phenolics: Antimicrobial Properties and Mechanisms of Action Against Severe Human Pathogens", NUTRITION AND CANCER, vol. 54, no. 1, 2006, pages 18 - 32, XP003023871, DOI: 10.1207/s15327914nc5401_4
RIITTA PUUPPONEN-PIMIALIISA NOHYNEKRIIKKA JUVONENTUIJA KÖSSÖPILAR TRUCHADOBENITA WESTERLUND-WIKSTROMTIINA LEPPANENEEVA MOILANENKIR: "Fermentation and dry fractionation increase bioactivity of cloudberry (Rubus chamaemorus)", FOOD CHEMISTRY, vol. 197, 2016, pages 950 - 958, XP029312168, ISSN: 0308-8146, Retrieved from the Internet DOI: 10.1016/j.foodchem.2015.11.061
PUUPPONEN-PIMIA, R.NOHYNEK, L.HARTMANN-SCHMIDLIN, S.KÄHKÖNEN, M.HEINONEN, M.MÄÄTTÄ-RIIHINEN, K.OKSMAN-CALDENTEY, K.: "Berry phenolics selectively inhibit the growth of intestinal pathogens", JOURNAL OF APPLIED MICROBIOLOGY, vol. 98, no. 4, 2005, pages 991 - 1000
SILVEIRA, M. H. L.AGUIAR, R. S.SIIKA-AHO, M.RAMOS, L. P.: "Assessment of the enzymatic hydrolysis profile of cellulosic substrates based on reducing sugar release", BIORESOUR. TECHNOL., 2014
TIKUNOV, Y.M., LAPTENOK, S., HALL, R.D: "MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data", METABOLOMICS, vol. 8, 2012, pages 714 - 718, XP035086048, Retrieved from the Internet DOI: 10.1007/s11306-011-0368-2
Attorney, Agent or Firm:
BOCO IP OY AB (FI)
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Claims:
Claims 1. A method for producing an antimicrobial extract from chicory biomass, characterized, in that the method comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme; d. incubating said aqueous chicory suspension and the enzyme mixture with mixing to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity. 2. The method according to claim 1, characterized in that a dry matter content of chicory suspension in step b. is 1 – 20 % w/v of chicory biomass, preferably 1 – 10 % w/v of chicory biomass, more preferably 3 – 7 % w/v of chicory biomass, most preferably 5 % w/v of chicory biomass. 3. The method according to claim 1 or 2, characterized in that the at least one enzyme is a food grade enzyme. 4. The method according to any one of the preceding claims, characterized in that the at least one enzyme is a hydrolytic enzyme selected from the group consisting of pectinase, xylanase, pectolyase, polygalacturonase, xylosidase, and xylanohydrolase. 5. The method according to any one of the preceding claims, characterized in that the amount of the enzyme is 1 – 10 mg/g dry matter of chicory biomass, preferably 2 – 8 mg/g dry matter of chicory biomass, more preferably 4 – 6 mg/g dry matter of chicory biomass, most preferably 5 mg/g dry matter of chicory biomass. 6. The method according to any one of the preceding claims, characterized in that the incubation in step d. is carried out for 6 – 48 hours, preferably for 10 – 40 hours, more preferably for 20 – 30 hours, most preferably for 24 hours.

7. The method according to any one of the preceding claims, characterized in that the incubation in step d. is carried out at a temperature of 20°C – 95°C, preferably at 30°C - 80°C, more preferably at 40°C – 60°C, most preferably at 50°C. 8. The method according to any one of the preceding claims, characterized in that the incubation in step d. is carried out at a pH of about pH 3.0 to about pH 10.0, preferably at about pH 4.5 to about pH 7.0, preferably at about pH 4.8 to about pH 5.2, more preferably at about pH 4.9 to about pH 5.1, most preferably at pH 5.0. 9. The method according to any one of the preceding claims, characterized in that the heat treatment in step e. is carried out by heating the enzymatically treated chicory suspension at a temperature of 80°C – 120 °C, preferably at 95°C - 100 °C, more preferably at 90°C – 100 °C, for at least 5 minutes. 10. The method according to any one of the preceding claims, characterized in that the antimicrobial activity is against a bacterium selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. 11. A chicory extract having antimicrobial activity obtainable with the method of any one of claims 1 – 10. 12. A chicory extract having antimicrobial activity, characterized in that the chicory extract: contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin and their oxalate and dihydro forms; has a pH above 3.9, preferably the pH is pH 4.5 – pH 5.5, more preferably pH 5.0; has the antimicrobial activity against a bacterium or bacteria selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. 13. A product comprising a chicory extract according to claim 12, characterized in that the product is a cosmetic product, hygiene product, health care product, or personal care product. 14. Use of a chicory extract with antimicrobial activity obtained by the method of any one of claims 1 to 10. 15. Use of a chicory extract of claim 12 as an antimicrobial agent in a cosmetic product, hygiene product, health care product and/or personal care product.

Description:
METHOD OF PRODUCING ANTIMICROBIAL EXTRACT FROM CHICORY FIELD OF THE DISCLOSURE The present disclosure relates to a method for producing an antimicrobial extract from chicory biomass. The disclosure further concerns a chicory extract having antimicrobial activity obtainable with said method. The present disclosure further concerns products comprising the obtained chicory extract having antimicrobial activity. Furthermore, the present disclosure concerns the use of said chicory extract in cosmetic products, hygiene products, personal care products or health care products. BACKGROUND OF THE DISCLOSURE Currently, synthetic antibacterial compounds such as parabens, which are considered harmful, are used in cosmetic industry. Parabens will most likely be forbidden in the future. Therefore, cosmetic industry seeks for new, safe antimicrobial compounds to be used as antimicrobial agents. Chicory [Cichorium intybus L. (Asteraceae)] is a perennial woody herb, whose Greek and Latin name derives from the words “field” (Cichorium), “to cut” and “tubus” to indicate the hollow stem (intybus). Chicory root contains up to 40 % inulin, which is a fructose polymer with a terminal glucose residue and is considered as a dietary fibre with negligible effect on human blood glucose levels. Chicory is cultivated for inulin industrial use 1.8 million tons annually in EU. Chicory can be divided into four main varieties or cultigroups according to their use (1) “industrial” or “root” chicory, produces the taproot as a coffee substitute or for inulin extraction; (2) “Belgian endive” or “witloof” chicory is commonly cultivated around Europe as industrial chicory for etiolated buds (chicons) by forcing; (3) “leaf” chicory or “radicchio” is used as fresh or cooked vegetables; and (4) “forage” chicory, initially derived from wild chicory, has been used since the mid-1970s to intensify herbage obtainability in perennial pastures for livestock. Besides inulin, chicory produces various secondary metabolites, which have shown to possess bioactive properties. These include sesquiterpenes, polyphenols and other phenolic compounds and their derivatives (Bogdanović et al. 2019). In the chicory industry a side-stream is formed after inulin is extracted from chicory. The side- stream is currently treated as waste. Patent publication CN 107349124 discloses a preservative for cosmetics made of the combination of three different plant-derived compounds, one of which is a chicory sesquiterpene lactone. Despite the advances in the technology to produce antimicrobial compounds for industry there remains a need of improved methods and products. BRIEF DESCRIPTION OF THE DISCLOSURE An object of the present disclosure is to provide a method and product which overcome the above problems related to the presently used methods for producing antimicrobial compounds. The object of the disclosure is achieved by a method and product which are characterized by what is stated in the independent claims. The preferred embodiments of the disclosure are disclosed in the dependent claims. The present inventors surprisingly found that when the chicory biomass is treated with an enzyme or a mixture of enzymes a chicory extract with antimicrobial activity i.e. with increased antibacterial activity against human skin pathogenic bacteria such as Staphylococcus aureus and/or Pseudomonas aeruginosa is obtained. The chicory extract obtained with a present method can be used as an antimicrobial agent in cosmetic products, hygiene products, and/or personal care products. The chicory extract obtained with the present method can also be used in health-care products. An advantage of the present invention is that new natural and safe antimicrobial compounds for cosmetic and health-care purposes are obtained from cheap and underutilized industrial waste stream. Another advantage of the present invention is that after application of an enzyme or enzymes, such as pectinase and xylanase, on chicory biomass, the antibacterial activity towards typical human skin pathogenic bacteria, such as Pseudomonas aeruginosa, and Staphylococcus aureus is observed. It was surprisingly found out that the extract was not inhibiting the growth of beneficial skin microbes such as lactobacilli. Enzyme treatment releases antimicrobial compounds from chicory matrix. The compounds displaying antimicrobial properties are plant secondary compounds, such as phenolic or sesquiterpene compounds. The present inventors observed that antimicrobial activity was assessed in cosmetic formula challenge test and the antimicrobial activity was preserved and functional in the formula of the present disclosure. BRIED DESCRIPTION OF THE DRAWINGS In the following the disclosure is described in greater detail by means of preferred embodiments of the invention with reference to the accompanying figures. FIG. 1 illustrates amounts of sesquiterpene lactones in dried root of industrial chicory after inulin extraction before (3D no enzyme) and after enzymatic treatment (3A pectinase, xylanase). Sesquiterpene lactones are selected from the group consisting of dihydrolactucin, lactucin, lactucin 15-oxalate, 8-deoxylactucin, dihydro 8-deoxylactucin, dihydro lactucopicrin, lactucopicrin, and lactucopicrin 15-oxalate. FIG.2 illustrates the amount of chlorogenic acid in dried root of industrial chicory after inulin extraction before (3D no enzyme) and after enzymatic treatment (3A pectinase, xylanase). FIG. 3 illustrates LC-MS chromatograms of chicory sample before (upper panel) treatment with pectinase and xylanase, and after (lower panel) treatment with pectinase and xylanase. Indicated with arrows and numbers are the chromatographic peaks that were subjected to multistage mass spectrometry (MSn) analyses. FIG. 4A illustrates antimicrobial activity of the enzyme treated chicory extract towards skin pathogen Staphylococcus aureus (VTT E-70045). The tested samples: Enzyme-treated chicory (3_3A) in 100 µl of acetic acid buffer; Non-treated chicory solution (3_3D) in 100 µl of acetic acid buffer. Commercial antibiotic, chloramphenicol 50 µg/ml with 100 µl of acetic acid buffer, was used as a comparative sample. Microbe control was incubated with 100 µl of acetic acid buffer. Enzyme-treated chicory (3_3A) sample showed growth inhibiting effect on S. aureus. FIG. 4B illustrates antimicrobial activity of the enzyme treated chicory extract towards skin pathogen Pseudomonas aeruginosa (VTT E-84219). The tested samples: Enzyme-treated chicory (3_3A) in 100 µl of acetic acid buffer; Non-treated chicory solution (3_3D) in 100 µl of acetic acid buffer. Commercial antibiotic, chloramphenicol 50 µg/ml with 100 µl of acetic acid buffer, was used as a comparative sample. Microbe control was incubated with 100 µl of acetic acid buffer. Enzyme-treated chicory (3_3A) sample showed a remarkable antimicrobial effect against Gram negative P. aeruginosa. FIG. 5 illustrates antimicrobial activity of the enzyme treated chicory extract towards skin beneficial bacteria Streptococcus thermophilus (VTT E-96665). The tested samples: Enzyme- treated chicory (3_3A) in 100 µl of acetic acid buffer; Non-treated chicory solution (3_3D) in 100 µl of acetic acid buffer. Commercial antibiotic, chloramphenicol 25 µg/ml with 100 µl of acetic acid buffer, was used as a comparative sample. Microbe control was incubated with 100 µl of acetic acid buffer. Enzyme control MIX A (pectinase + xylanase 10 mg/ml). There was no effect of 3_3A towards Streptococcus thermophilus. FIG. 6 illustrates antimicrobial activity of the enzyme treated chicory extract towards skin beneficial bacteria Lactobacillus rhamnosus (VTT GG E-96666). The tested samples: Enzyme- treated chicory (3_3A) in 100 µl of acetic acid buffer; Non-treated chicory solution (3_3D) in 100 µl of acetic acid buffer. Commercial antibiotic, chloramphenicol 25 ug/ml with 100 µl of acetic acid buffer, was used as a comparative sample. Microbe control was incubated with 100 µl of acetic acid buffer. Enzyme control MIX A (pectinase + xylanase 10 mg/ml). There was no effect of 3_3A towards Lactobacillus rhamnosus. FIG. 7A illustrates microbial challenge test in standard cosmetic cream formula. An enzyme treated sample 3_3A was inserted to cosmetic formula (F) and the antimicrobial activity of the formula (F+4% 3_3A) was followed. During incubation period of four weeks, a clear antimicrobial activity of the sample 3_3A against Staphylococcus aureus was observed, showing better activity than commercial preservative (F+0.5% preservative) and non-enzymatically treated chicory solution (3_3D). FIG. 7B illustrates microbial challenge test in standard cosmetic cream formula. An enzyme treated sample 3_3A was inserted to cosmetic formula (F) and the antimicrobial activity of the formula was followed. During incubation period of four weeks, a clear antimicrobial activity of the sample 3_3A against Pseudomonas aeruginosa was observed, showing better activity than commercial preservative and non- enzymatically treated chicory solution 3_3D. DETAILED DESCRIPTION OF THE DISCLOSURE This disclosure describes a method for producing an antimicrobial extract from chicory biomass, wherein the method comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme; d. incubating said aqueous chicory suspension and the enzyme mixture with mixing to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity. Chicory, especially chicory biomass, is used for producing an antimicrobial chicory extract. A method for producing said extract comprises providing chicory, preferably chicory biomass, as a starting material. An example of chicory biomass is dried chicory, such as chicory root material from industrial chicory after inulin extraction. The chicory biomass is mixed with an aqueous medium to obtain an aqueous chicory suspension. An enzyme mixture comprising at least one enzyme, preferably selected from hydrolytic enzymes is added to the aqueous chicory suspension. Preferably, the enzyme treatment is carried out at a pH of about pH 4.5 to about pH 7.0, preferably at a temperature of between 20 – 95 °C, preferably for 6 – 48 hours to obtain an enzymatically treated chicory suspension. Enzyme dose may be determined based on protein content. In the end of the treatment enzymes are inactivated by heat treatment. Preferably, the heat treatment is carried out at a temperature of 80 to 120 °C for at least 5 minutes to obtain a heat-treated chicory suspension. Insoluble solids are separated from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity. The chicory extract obtained by the present method has antibacterial activity against human skin pathogenic bacteria such as Staphylococcus aureus and/or Pseudomonas aeruginosa. The extract does not inhibit the growth of beneficial skin microbes such as lactobacilli. The chicory extract may contain one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin and their oxalate and dihydro forms. The extract may have a pH above 3.9, such as from pH 3.9 to pH 6.0. Preferably the pH is pH 4.5 – pH 5.5, more preferably pH 5.0 or about pH 5.0. The chicory extract of the present disclosure may be used as an antimicrobial agent in cosmetic products, hygiene products and/or personal care products. The chicory extract obtained with the present method may also be used in health-care products. The present method for producing an antimicrobial extract from chicory biomass comprises the following step: providing chicory biomass. The term “biomass” commonly refers to any substance of biotic origin. Biomass may be expressed as fresh weight, or as dry weight (excluding water contained in organisms). Plant biomass is referred as renewable organic material that comes from plants. The chicory biomass may be any chicory plant material or originate from any chicory plant material such as from chicory plant or from chicory processing or from chicory processing side streams. Any form of chicory may be used, such as fresh or dry chicory. In an embodiment chicory plant is Cichorium intybus L. var sativum. In an embodiment the chicory biomass is in a form of chicory powder. In an embodiment dried chicory is used. For example, dried chicory may be dried root of industrial chicory after inulin extraction. The present method also comprises the following step: preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension. In an embodiment a pH of the aqueous chicory suspension is adjusted to the range between pH 4.5 and pH 5.5, preferably a pH of the aqueous chicory suspension is adjusted to pH 5.0, or about pH 5.0. The pH may be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5, or in the range defined by any two of these values. A dry matter content of chicory suspension in step b. may be 1 – 20 % w/v of chicory biomass preferably 1 – 10 % w/v of chicory biomass, more preferably 3 – 7 % w/v of chicory biomass, most preferably 5 % w/v of chicory biomass. The dry matter content of chicory suspension in step b. may be such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 % w/v of chicory biomass, or in the range defined by any two of these values. The method also comprises the following step: adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme. In an embodiment the at least one enzyme is a food grade enzyme. In an embodiment two enzymes are used. Preferably, the two enzymes are food grade enzymes. The enzyme or enzymes in an enzyme mixture are typically hydrolytic enzymes. Hydrolytic enzyme is any enzyme that catalyses the hydrolysis of a chemical bond. Hydrolytic enzymes include for example proteases, lipase, phosphatase and esterases. In an embodiment the at least one enzyme is a hydrolytic enzyme selected from the group consisting of pectinase, xylanase, pectolyase, polygalacturonase, xylosidase, and xylanohydrolase. In an embodiment the at least two enzymes are selected from the group consisting of pectinase, xylanase, pectolyase, polygalacturonase, xylosidase, and xylanohydrolase. In an embodiment the enzyme or enzymes is selected from the group consisting of pectinase, xylanase, β-glucosidase, inulinase, cellulase, and protease. In an embodiment the at least one enzyme is selected from the group consisting of inulinase pectinase, cellulase, β-glucosidase, xylanase, and esterase. Said enzymes can be used alone or in any combinations with each other. The dosage of the enzyme or enzymes can be selected from the dosages suitable for each purpose, as known to a person skilled in the art. In a preferred embodiment of the present method the enzyme is selected from the group consisting of pectinase, xylanase, beta-glucosidase, inulinase, cellulase, and protease or any combination thereof. In another preferred embodiment of the present method the enzyme is selected from the group consisting of pectinase, xylanase and β-glucosidase or any combination thereof. In a more preferred embodiment of the present method the enzyme is mixture of pectinase and xylanase. One suitable inulinase is Fructozyme (Novozymes). One suitable cellulase is Cellic CTec2 (Novozymes). One suitable β-glucosidase is Novozym 188 (Novozymes). One suitable xylanase is Depol 40L (Biocatalysts Ltd.). One suitable esterase is ferulic acid esterase Depol 740L. It may also be possible to obtain suitable enzymes from microbial sources by biotechnical screening methods or by improving existing enzymes by protein engineering. In further embodiments of the present method, two, three, four, five, or six enzymes are used. Pectinolytic enzymes are typically used in juice processing industry to ameliorate the juice extraction. Most of these commercial pectinase preparations are mixtures of endoacting (carbohydrate backbone cleaving) and exoacting (carbohydrate side-chain cleaving) pectinases together with cellulases and hemicellulases. Enzymatic treatment enhances the extractability of phenolic compounds from plant cell wall matrix. In addition, exoacting properties of these enzymes are particularly interesting with potential effects on the chemistry of phenolic glycosides. Typically, the amount of the enzyme or enzymes used in the present method is from 0.1 – 10 mg/g dry matter of chicory biomass, preferably 1 – 8 mg/g dry matter of chicory biomass, more preferably 2 – 6 mg/g dry matter of chicory biomass, more preferably 4 – 6 mg/g dry matter of chicory biomass, most preferably 5 mg/g dry matter of chicory biomass. The amount of the enzyme may be such as 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/g dry matter of chicory biomass, or in the range defined by any two of these values. Definitions and assay conditions for activity units in enzyme preparations may vary between enzyme providers. Substrates used in the enzyme assays do not generally totally represent the chicory biomass used and may also vary. Enzyme preparations are mixtures of several molecules with different enzyme activities. Because of these reasons, selection of the activity of one single type of protein is arbitrary and may not describe properly the combinatory effect of the whole activity spectrum present in a preparation. For these reasons, the protein content (such as mg/g dry matter of chicory biomass) can be seen as most reliable basis for dosing the preparation. Enzyme dose may be calculated based on the protein content. The method also comprises the following step: incubating said aqueous chicory suspension and the enzyme mixture with mixing to obtain an enzymatically treated chicory suspension. The incubation the aqueous chicory suspension and the enzyme mixture in step d. may be carried out for 6 – 48 hours, preferably for 10 – 40 hours, more preferably for 20 – 30 hours, most preferably for 24 hours. The incubation in step d. may be carried out 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours, or for a time in the range defined by any two of these values. The incubation in step d. may be carried out at a temperature of 20°C – 95°C, preferably at a temperature of 30°C - 80°C, more preferably at 40°C – 60°C, most preferably at 45°C – 55°C, such as at 50°C or at about 50°C. The incubation may for example be carried out at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C or at a temperature in the range defined by any two of these values. In an embodiment the incubation of the aqueous chicory suspension and the enzyme mixture is carried out at a temperature of 45°C – 55°C, such as at 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C, or at a temperature in the range defined by any two of these values. The incubation may be carried out at a pH of about pH 3.0 to about pH 10.0, preferably at a pH of about pH 4.5 to about pH 7.0, preferably at a pH of about pH 4.5 to about pH 5.5, preferably at a pH of about pH 4.8 to about pH 5.2, more preferably at a pH from about pH 4.9 to about pH 5.1, most preferably at pH 5.0. The incubation may be carried out at a pH 3.0, 3.5, 4.0, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 9.5 or 10.0 or in the range defined by any two of these values. The amount of an enzyme or enzymes, incubation time, and incubation temperature may be combined in a suitable way. The method also comprises the following step: subjecting the enzymatically treated chicory suspension to heat treatment to obtain a heat-treated chicory suspension. The heat treatment may be carried out by heating the enzymatically treated chicory suspension at a temperature of 80°C to 120°C, preferably at 95°C - 100°C, more preferably at 90°C - 100°C for at least 5 minutes. The heat treatment may be carried out at a temperature of 80°C, 81°C, 82°C, 83°C , 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C, 110°C, 111°C, 112°C, 113°C, 114°C, 115°C, 116°C, 117°C, 118°C, 119°C or 120°C, or in the range defined by any two of these values. In an embodiment the heat treatment is carried out from 5 minutes to 30 minutes. The heat treatment may be carried out for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 minutes, or for a time in the range defined by any two of these values. In a preferred embodiment the heat treatment is carried out for 15 minutes at a temperature of 90°C to 100°C. The method also comprises the following step: separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity. The separation may be carried out by centrifugation or filtration, preferably by filtration. The antimicrobial activity may be against a bacterium or bacteria selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 1 – 20 % w/v of chicory biomass, preferably 1 – 10 % w/v of chicory biomass, more preferably 3 – 7 % w/v of chicory biomass, most preferably 5 % w/v of chicory biomass; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme selected from hydrolytic enzymes; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 4.5 to about pH 7.0, preferably at about pH 4.5 to about pH 5.5, more preferably at about pH 4.9 to about pH 5.1, most preferably at pH 5.0, at a temperature of between 20 – 95 °C, preferably between 40 – 60 °C, , most preferably at 50 °C, for 6 – 48 hours, preferably for 10 – 40 hours, more preferably for 20 – 30 hours, most preferably for 24 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 80 to 120 °C, preferably at 95 to 100°C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 1 – 20 % w/v of chicory biomass; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme selected from hydrolytic enzymes; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 4.5 to about pH 7.0, at a temperature of between 20 – 95 °C, for 6 – 48 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 80 to 120 °C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 1 – 10 % w/v of chicory biomass, preferably 3 – 7 % w/v of chicory biomass, more preferably 5 % w/v of chicory biomass; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme selected from hydrolytic enzymes; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 4.5 to about pH 5.5, preferably at about pH 4.8 to about pH 5.2, more preferably at about pH 4.9 to about pH 5.1, most preferably at pH 5.0, at a temperature of between 40 – 60 °C, , more preferably at 50 °C, for 6 – 48 hours, preferably for 10 – 40 hours, more preferably for 20 – 30 hours, most preferably for 24 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 95 to 100 °C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus, and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 5 % w/v of chicory biomass; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme selected from hydrolytic enzymes; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 5.0, at a temperature of 40 - 60°C, more preferably at 50 °C, for 20 – 30 hours, more preferably for 24 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 95 to 100 °C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus, and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 5 % w/v of chicory powder; c. adding to said aqueous chicory suspension an enzyme mixture comprising at least one enzyme selected from pectinase, xylanase, β-glucosidase, inulinase, cellulase and protease; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 4.5 to pH 5.5, preferably at about pH 5.0, at a temperature of 40 - 60°C, preferably at 45 – 55 °C, more preferably at 50 °C, for 20 – 30 hours, more preferably for 24 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 95 to 100 °C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus, and Pseudomonas aeruginosa. In an embodiment a method for producing an antimicrobial extract from chicory biomass, comprises the steps of a. providing chicory biomass; b. preparing a chicory suspension by mixing said chicory biomass and aqueous medium to obtain an aqueous chicory suspension with a dry matter content of 5 % w/v of chicory powder; c. adding to said aqueous chicory suspension an enzyme mixture comprising pectinase and xylanase; d. incubating said aqueous chicory suspension with mixing at a pH of about pH 4.5 to pH 5.5, preferably at about pH 5.0, at a temperature of 40 - 60°C, preferably at 48 – 52 °C, more preferably at 50 °C, for 20 – 30 hours, more preferably for 24 hours to obtain an enzymatically treated chicory suspension; e. subjecting the enzymatically treated chicory suspension to heat treatment at a temperature of 95 to 100 °C for at least 5 minutes to obtain a heat-treated chicory suspension; f. separating insoluble solids from the heat-treated chicory suspension to obtain a chicory extract with antimicrobial activity against a bacterium selected from the group consisting of Staphylococcus aureus, and Pseudomonas aeruginosa. The disclosure also describes a chicory extract having antimicrobial activity obtainable with the presently described method. An aspect of the present disclosure is a use of chicory extract with antimicrobial activity obtained by the presently disclosed method. The use or chicory extract may relate to therapeutic or non- therapeutic use. An aspect of the present disclosure is a use of an extract obtained by the presently disclosed method as an antimicrobial agent. The extract may be used in a cosmetic product, in a hygiene product, in a health care product, or in a personal care product. The disclosure also describes a chicory extract having antimicrobial activity, wherein the chicory extract: contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin, and their oxalate and dihydro forms; has a pH above 3.9, preferably the pH is pH 4.5 – pH 5.5, more preferably pH 5.0; has the antimicrobial activity against a bacterium or bacteria selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In an embodiment the chicory extract contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin, dihydrolactucin, dihydro 8-deoxylactucin, dihydro lactucopicrin, lactucin 15-oxalate, and lactucopicrin 15-oxalate. In an embodiment the pH of the chicory extract having antimicrobial activity is pH 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5, or in the range defined by any two of these values. The pH of the chicory extract may be from pH 3.9 to pH 6.0. The pH may pH 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.55.6, 5.7, 5.8, 5.9, or 6.0, or in the range defined by any two of these values. In an embodiment the pH is from pH 3.9 to pH 6.0. In an embodiment the chicory extract having antimicrobial activity contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8- deoxylactucin, lactucopicrin, dihydrolactucin, dihydro 8-deoxylactucin, dihydro lactucopicrin, lactucin 15-oxalate, and lactucopicrin 15-oxalate; has a pH above 3.9, preferably the pH is 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or in the range defined by any two of these values; and has the antimicrobial activity against a bacterium or bacteria selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In an embodiment the chicory extract having antimicrobial activity against Staphylococcus aureus contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin, dihydrolactucin, dihydro 8- deoxylactucin, dihydro lactucopicrin, lactucin 15-oxalate and lactucopicrin 15-oxalate; and has a pH above 3.9, preferably the pH is 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or in the range defined by any two of these values. In an embodiment the chicory extract having antimicrobial activity against Pseudomonas aeruginosa contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin, dihydrolactucin, dihydro 8-deoxylactucin, dihydro lactucopicrin, lactucin 15-oxalate and lactucopicrin 15-oxalate; and has a pH above 3.9, preferably the pH is 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or in the range defined by any two of these values. In an embodiment the disclosure describes a chicory extract having antimicrobial activity, wherein the chicory extract: contains one or more chicory sesquiterpenes selected from the group consisting of lactucin, 8-deoxylactucin, lactucopicrin, and their oxalate and dihydro forms; has a pH from 3.9 to pH 6.0, preferably the pH is from pH 4.5 to pH 5.5, more preferably pH 5.0; has the antimicrobial activity against a bacterium or bacteria selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. The disclosure also describes a product comprising an extract having antimicrobial activity obtainable with the presently described method, wherein the product is a cosmetic product, hygiene product, health care product or personal care product. The chicory extract may be added to a product, such as cosmetic product or personal care product, for example to a cosmetic cream in 4 % (v/v) – 10 % (v/v) concentration, such as in a concentration of 4 % (v/v), 5 % (v/v), 6 % (v/v), 7 % (v/v), 8 % (v/v), 9 % (v/v), or 10 % (v/v), or in a concentration in the range defined by any two of these values. In an embodiment the pH of the product comprising the chicory extract is from pH 3.9 to pH 6.0, such as pH 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, or in the range defined by any two of these values. The cosmetic product may be selected from the group consisting of a skin care product, such as cleanser, toner, facial mask, moisturizer, cosmetic cream, sunscreen and several other product types, and make up product, such as eye liner, lipstick, lip gloss and several other product types. A cosmetic cream may comprise an oil phase and a water phase. The oil phase may comprise components such as polyglyceryl-3-methylglucose distearate, glycerol stearate, cetearyl alcohol, caprylic/capric triglyceride and/or oeyl erucate. The water phase may comprise water and glycerin. In an embodiment the chicory extract is sterilized, such as filter sterilized, and diluted in glycerin before adding to a skin care product, such as a cream formula. In an embodiment a cream formula may comprise 78% aqua, and 3% glycerin as a water base, as well as 6% caprylic/capric triglyceride, 4% oleyl erucate, 3% glycerol stearate, 3% cetearyl alcohol, and 3% polyglyceryl-3-methylglucose distearate as an oil base. Choosing a formulation of a cream is known to a skilled person in the art. In an embodiment a cream formula comprises about 78% aqua, and about 3% glycerin as a water base, as well as about 6% caprylic/capric triglyceride, about 4% oleyl erucate, about 3% glycerol stearate, about 3% cetearyl alcohol, and about 3% polyglyceryl-3-methylglucose distearate as an oil base. The personal care product may be selected from the group consisting of cleansing pads, colognes, cotton swabs, cotton pads, deodorant, facial tissue, hair clippers, lip balm, lotion, hand soap, facial cleanser, body wash, nail files, pomade, perfumes, razors, shaving cream, moisturizer, baby powder, toilet paper, toothpaste, facial treatments, wet wipes, towels, and shampoo. An aspect of the present disclosure is a use of an extract obtained in the present method as an antimicrobial agent in a cosmetic product, in a hygiene product, in a health care product, or in a personal care product. In an embodiment the chicory extract is for use as an antimicrobial agent in a therapeutic application. In another embodiment the chicory extract is for use as an antimicrobial agent in a non-therapeutic application. The chicory extract obtained in the present method may be used for example to replace preservatives or synthetic antimicrobial compounds, such as parabens or phenoxyethanol, in a cosmetic product either entirely or partially. It is apparent to a person skilled in the art that as technology advances, the basic idea of the invention can be implemented in various ways. The invention and its embodiments are therefore not restricted to the above examples, but they may vary within the scope of the claims. EXAMPLES Materials and Methods Chicory material A chicory root sample was received from ILVO (Flanders Research Institute for Agriculture, Fisheries and Food, Belgium). The sample consisted of industrial chicory after inulin extraction. The sample composition is shown in Table 1. Table 1. Composition of the chicory sample. Results are presented based on fresh weight. NA: no data available Enzymatic treatment Samples were suspended into 0.05 M acetate buffer (pH 5.0) to dry matter content of 5 % w/v. The total volume of the suspension was 20 ml. Samples were incubated with mixing at 50 °C for 24 h in temperature cabinet (Infors HT Minitron). Altogether six enzymes were used alone or in combinations: inulinase (Fructozyme L, Novozymes); pectinase (Pectinex Smash, Novozymes); cellulase (Cellic CTec2, Novozymes); β-glucosidase (Novozym 188, Novozymes); xylanase (Depol 40L, Biocatalysts); and esterase (Depol 740L, ferulic acid esterase). Enzyme dose was calculated based on the protein content. In the end of the treatment, enzymes were inactivated by boiling the sample for 15 min. After inactivation, the samples were centrifuged (4000 rpm, 15 min), supernatants collected and provided for further analyses. Samples were stored at -20 °C before bioactivity measurements. Determination of reducing sugars The sugars released in enzymatic hydrolysis experiments were analysed by reducing sugar assay according to the dinitrosalicylic acid (DNS) colorimetric method in 96-well format (Silveira et al. 2014). Antimicrobial activity assay Antimicrobial activities of the samples were evaluated from enzyme treated samples using the liquid culture method (Nohynek et al. 2006) with modification of using liquid microbial cultures of 1 ml (Puupponen-Pimiä et al., 2016). Microbial strains used in the analysis were Staphylococcus aureus VTT E-70045 (ATCC 6538), Pseudomonas aeruginosa VTT E-84219 (ATCC 15692), Lactobacillus rhamnosus GG VTT E-96666 (ATCC 53103) and Streptococcus thermophilus VTT E-96665 (ATCC 19258). S. aureus and P. aeruginosa were cultured aerobically at 37 °C in Nutrient broth (Difco) with agitation (150 rpm). L. rhamnosus and S. thermophilus were cultured in MRS broth (De Man Rogosa Sharpe; Oxoid, Basingstoke, UK) at 37 °C without agitation. Microbial stock cultures were maintained frozen at –80 °C. For experimental use the microbial cultures were transferred onto solid media and incubated for 1–2 days as described above for each strain. The colonies were introduced into liquid media, incubated for 20–24 h, and used as the source of inoculum for antimicrobial activity analysis. Microbial sample with only enzyme in 100 µl of acetic acid buffer was used as a positive growth control, and sample with antibiotic compound (chloramphenicol) with 100 µl of acetic acid buffer was used as a negative control for microbial growth. The inhibitory effects of samples in 100 µl of acetic acid buffer on the microbes were evaluated by comparing the control growth curves with those obtained from cultures with samples. Chemical analyses Enzyme-treated chicory sample treated with pectinase and xylanase, and its respective non- treated control was analysed by untargeted metabolomics analyses. Untargeted LC-MS analysis was performed to detect sesquiterpene lactones (SL) and phenolics in the samples after enzyme treatment. Next to that, untargeted GC-MS analysis was performed to detect possible differences in more hydrophobic compounds in the extract. Analyses were performed essentially as described in Cankar et al. (2021). Briefly, supernatants from treated chicory samples were frozen and 300 µl of sample was extracted with 700 µl of methanol (100% MeOH + 0.1% formic acid). The mixture was incubated for 15 minutes in a sonicator (Branson 3510) to facilitate extraction. The cell debris was separated from the extract by centrifugation (10 min, 15000 rpm). Before analyses, 100 µl of the extract was transferred to a glass vial and analysed on the Orbitrap_LC-MS in positive mode. For data processing, the raw data from the LC‐MS were processed using MetAlign software (Lommen, 2009) in order to correct for background and noise signals and to perform alignment of the chromatographic peaks from the different samples. Using MetAlign Output Transformer (Houshyani et al. 2012) the MetAlign output was subsequently cleaned up by removing mass data showing values lower than the detection threshold (s/n ≥ 3). Using MSClust software (Tikunov et al. 2012), the remaining mass peaks were clustered using information on their original retention time and their peak intensity pattern across all samples, forming a mass cluster or centrotype. The centrotype represents the original metabolite with a relative intensity per sample. Cosmetic formula challenge test The challenge test is based on the method, with defined concentrations of commercial preservative and chicory by-product sample 3_3A (enzyme-treated) and 3_3D (non-treated) are added in the formula, and their antimicrobial activity is tested against selected microbes according to the protocol of ISO 11930 Preservative Effectiveness Test, with some modifications. As a positive control, commercial preservative Euxyl®PE 9010 (phenoxyethanol & ethylhexylglycerin) at concentration of 0.5 % in formula was used. Chicory extracts were added in 4 % (v/v) concentration. Cosmetic cream formula was prepared according to Table 2, as follows: Oil (phase A) and water (phase B) were weighed and both phases were heated separately until they reached 80 °C. Oil phase was gently added into water phase with continuous stirring using sterilized milk foamer (10 min). After the emulsion became viscous and homogenous, it was cooled to 40 °C. Subsequently, extract diluted in glycerin (1:1) was added by stirring. Final pH was adjusted to pH 5.5. Samples were let to set overnight before inoculation of microbes and adding of commercial preservative control Euxyl PE 9010. Table 2. Composition of standard cream formula. Phase Compound INCI name Amount (%) A TEGO CARE 450 Polyglyceryl-3-methylglucose distearate 3 CITHROL GMS 40 Glycerol stearate 3 TEGO ARGANOL 1618 Cetearyl alcohol 3 BERGABEST MCT-OIL 60/40 Caprylic/capric triglyceride 6 TEGOSOFT OER Oleyl erucate 4 B WATER Aqua 78 GLYCERIN Glycerin 3 Microbes used for challenge test were Staphylococcus aureus (VTT E-70045, ATCC 6538) and Pseudomonas aeruginosa (VTT E-96728, ATCC 9027) in levels of 1x10 5 - 1x10 6 cfu /g formula. First, 10 g of formula was aseptically weight into sterile 50 mL falcon tube. Chicory extracts were filter sterilized and diluted in glycerin (1:1) before adding to the cream formula aseptically at a final concentration of 4 % of glycerin-diluted extract in the cream formula sample. As a control, cream formula without preservative or chicory extract was used. Microbial cultures were initiated by incubating at 37°C for 18 h to 24 h on tryptone soy agar (TSA) and they were sub-cultured once before starting of the experiment. Bacterial biomass was collected by taking a loopful from the agar plate into 5 ml of Peptone-Saline (PS) solution in 20 ml tube with glass beads and the tube was subsequently vortexed to break down cell aggregates. The density of the suspension was adjusted with PS to Mac Farland 0.5 using densitometer (corresponding to appr. 10 7 -10 8 cfu/ml). Each microbial inoculum with MF 0.5 were added into formula with 100 µl by vortexing. Euxyl 9010 represented commercial preservative and was added (0.5 ml) into formula samples of positive controls. Formula samples were incubated at 25 o C, and sampling was performed once a week until four weeks. Each sample (0.5 g of the formula controls and formula inoculated with microbes) was suspended to 4.5 ml of PS and serial dilutions were made for microbial counts on the plates. Microbial plates were incubated at 37°C and colonies were counted after 24 h, 48 h and 72 h. Results and Discussion Composition of chicory samples The nutrient composition of the chicory sample is presented in Table 1 and amounts of selected carbohydrates, phenolic compounds and sesquiterpene lactones in the sample are shown in Table 3. Table 3. Amounts of selected carbohydrates, phenolic compounds and sesquiterpene lactones in the chicory biomass. Dry matter was measured by weighing. Table 4. Activity profiles and protein content (mg/ml) of used enzymes. Activities are expressed as nkat/ml, except cellulase activity as FPU/ml, and assayed in pH 5, except inulinase in pH 6. *Bio Rad Lowry, after acetone precipitation a) Ghose (1987) 12 b ) IUPAC (1987) 13 c ) Bailey and Nevalainen (1981) 14 d ) Bailey et al. (1992) 15 e ) Bailey and Pessa (1989) 16 g ) Abu El-souond et al. (2014) 17 h ) Lowry et al. (1951) 18 Enzymatic treatment and antimicrobial activity Evaluation of different enzymes and enzyme combinations on release of sugars and antimicrobial activity were screened. Activity profiles of the used enzyme preparations, which are mixtures of different hydrolytic enzymes are shown in Table 4. Enzymes acting on hemicellulose (xylan) and pectin released largest amounts of hydrolysis products, measured as reducing sugars, whereas glucanolytic (cellulase and β-glucosidase), esterase and inulinase preparations alone had minor hydrolytic effects (Table 5). The preparations and especially their combinations with highest hydrolytic effect (pectinase and xylanase) showed also highest antimicrobial properties which resulted in more detailed studies using the combinations of the preparations (Table 6). Since the antimicrobial effects are most likely not due to pectic or hemicellulosic sugars, it is evident that the use of these enzymes facilitates the release of more complex organic molecules, responsible of antimicrobial characteristics. The mechanisms for this are not known, but they may occur due to the hydrolytic degradation of physical solid matrix of the substrate, resulting in the release of the actual bioactive compounds, or maybe partly due to still unknown minor side activities in the preparations, capable of releasing active components from chicory material by direct enzymatic action. Antimicrobial activity was tested in liquid cultures of selected microbial strains using chicory extracts. Cichorium sp. are known to possess antimicrobial activity. In this study, the industrial side stream of chicory was assessed with the aim to upgrade the fraction which remains after inulin extraction. In addition, the possibility to enhance the bioactivity by enzyme treatment was studied. Earlier it has been shown that application of cell wall degrading enzymes can increase the antibacterial activity of various bilberry side streams. Selected enzymes were initially tested with the concentration 50 mg/g (dosing according to protein content) and antimicrobial activity was assessed against Staphylococcus aureus with two sample concentrations. A clear growth inhibition with several enzyme treatments was shown, however, with that dosing, also some of the enzymes themselves displayed antimicrobial activity. Thus, in the next step the level of the enzyme was significantly reduced, to a concentration 5 mg/g protein. The results of the antimicrobial assessment are shown in Table 5. Table 5. Chicory sample treated with individual enzymes or enzyme mixtures (applied in the level of 5 mg/g protein), total reducing sugars (DNS) and assessed for antimicrobial activity against S. aureus. No inhibitory effect was observed with enzyme preparations, only. 1Levels of sample without treatment and enzyme only are subtracted. - no inhibitory effect; + weak inhibition; ++ clear inhibition; +++ strong inhibition; ++++very strong inhibition. The best growth inhibition against S. aureus was obtained with treatment combining pectinase, xylanase and β-glucosidase (Table 5). However, as β-glucosidase treatment alone did not have inhibitory effect towards S. aureus, in the following experiments the combination of pectinase and xylanase, without β-glucosidase was also assessed. In addition to S. aureus, antimicrobial activity towards other typical skin -related microbe Pseudomonas aeruginosa was studied. Results are shown in Table 6. Table 6. Chicory sample treated with enzyme mixture (applied in the level of 5 mg/g protein) and assessed for antimicrobial activity against S. aureus, and P. aeruginosa. No inhibitory effect was observed with enzyme preparations, only. 1Levels of sample without treatment and enzyme only are subtracted. - no inhibitory effect; + weak inhibition; ++ clear inhibition; +++ strong inhibition; ++++very strong inhibition. *growth retarding effect at 24 h, complete growth recovery at 48 h. As hypothesized, omission of β-glucosidase from the enzyme cocktail did not decrease the inhibitory effect of chicory and it was shown that pectinase and xylanase together were sufficient to result in strong inhibition (Table 6). A remarkable growth inhibition with chicory sample treated with pectinase and xylanase was observed against growth of Gram-negative P. aeruginosa. This sample was subjected to more detailed investigation for its remarkable effect on P. aeruginosa. Samples were taken along the liquid cultivation with the bacteria and both pH and chemical analyses were performed. It was observed that pH changed drastically immediately after start of the cultivation, while for controls and non-treated sample the pH remained in the level of the start or slightly higher (Table 7). Table 7. Changes in pH during P. aeruginosa cultivation of chicory sample. Finally, antimicrobial activity of the chicory sample after best enzyme treatment obtained earlier (Table 6), was assessed against skin beneficial bacteria. The results are presented in Table 8. Table 8. Chicory sample treated with combination of pectinase and xylanase (both applied in the level of 5 mg/g protein) and assessed for antimicrobial activity against Lactobacillus rhamnosus E-96666 and Streptococcus thermophilus E-96665. No inhibitory effect was observed with enzyme preparations, only. - no inhibitory effect; + weak inhibition; ++ clear inhibition; +++ strong inhibition; ++++very strong inhibition. Analyses of terpenes and polyphenols Enzyme-treated chicory sample and its non-treated control were subjected to chemical analyses for chicory sesquiterpenes and polyphenols (FIG. 1, FIG. 2). Lactucin, dihydrolactucin, 8-deoxylactucin, lactucopicrin, and dihydro-8-deoxylactucin were found in the samples and upon enzyme treatment the dihydro-SLs were increased. Chlorogenic acid concentration decreased after enzyme treatment (FIG. 1, FIG. 2). Upon the enzyme treatment a complex mixture of compounds was detected (FIG. 3). Several of these compounds had the same base peak mass, indicating structural similarity among them. To identify compounds, an untargeted metabolomics and MSMS were applied. Untargeted metabolomics analyses revealed a number of other unique compounds in this sample and the most differentially induced compounds were studied further. After MSn analyses fragmentation patterns and elemental composition was determined. In FIG. 3. LC-MS chromatogram of chicory sample before and after treatment with pectinase and xylanase is presented. Untargeted metabolomic profiling was chosen as a method to select metabolites that were specifically produced by the enzymatic treatment of the chicory pulp (after inulin extraction). The metabolomics pipeline resulted in an output of a total of 584 metabolites and their relative intensities in the samples. Metabolites present in chicory sample after treatment with pectinase + xylanase and absent in non-treated chicory sample and the other enzyme treated samples, were selected. This resulted in a list of 113 metabolites overrepresented in chicory sample after treatment with pectinase + xylanase. The most differential mass peaks in this sample compared to the other treatments were selected using Pearson's correlation with 0,98 correlation as cut off level. Successively, masses having a retention time lower than 5 minutes were excluded. Furthermore, to exclude low abundant metabolites, mass peaks with 10x lower mass peak area than the average mass peak area were omitted. Finally, to exclude artefacts from the data processing, the selected masses were checked for their occurrence in the raw data. From the selected masses, the corresponding mass chromatograms were studied and by comparisons with mass libraries, putative identifications were made. In the end, ten metabolites were selected for identification using MSMS (Table 9 and FIG. 3).

Table 9. MSn results per selected peak: The retention time, the selected mass for fragmentation, the characteristic mass fragments and the tentative peak identification. This analysis resulted in the putative identification of two sesquiterpene lactone degradation products, peak 1 and 2, eluting from the analytical column at resp. 12.64 and 15.9 minutes. Six compounds were putatively identified as amino group containing long chain alkanes. The similarity between the mass fragments of these 6 compounds was remarkable which indicates structural similarity between the molecules. The putative identification shows differences in nitrogen-composition. The MSMS degradation products of peaks 5, 8 and 9 show that the amino group is placed on the outside of molecule shown by the loss of the ammonia group during MSMS. The peaks 6, 7 and 10 are predicted to have multiple nitrogen atoms by the accurate mass estimation, however we did not observe loss of ammonia during MSn fragmentation. This could indicate that the nitrogen groups are located more strongly inside the molecule. The alkane chain is shown to be variable in size, for some molecules rather long alkane fragments are found, for example peak 6 shows a fragment of 10 carbons. Other show smaller alkane chain lengths-fragments. Differences in hydrogen addition to those chain fragments point out to more or less saturation of the chains. Peaks 3 and 4 did not result in an identification, even the molecular formula prediction could not be substantiated. The sesquiterpene lactone -like molecules could be breakdown products or modified chicory sesquiterpene lactones. The amino group containing long chain alkanes could originate from fatty acids from the chicory cell membranes or storage oils. Cosmetic formula challenge test Control of microbial growth in cosmetic products is crucial for maintenance of quality and safety. Microbial contamination in cosmetic product may lead to changes in the structure, e.g. viscocity and odour or colour. However, approximately 6% of the population is sensitized to the ingredients of cosmetics, especially to preservatives and fragrances (Canavez et al 2021). Preservatives are reported to cause e.g. skin irritation, erythema, contact allergy, contact sensitization and contact dermatitis (Halla et al. 2018). When chicory extracts were applied to standard cosmetic cream formula, as to replace a synthetic preservative, a strong reduction of common skin-related bacteria S. aureus and P. aeruginosa in challenge test was observed during the one month follow-up (FIG. 7). In FIG. 7. the results of microbial challenge test in standard cosmetic cream formula are presented. FIG. 7A) S. aureus, FIG. 7B) P. aeruginosa. Microbial numbers (cfu/g formula) were counted after 72 h incubation from sampling. Very strong reduction in S. aureus microbial load was observed already after 7 days of incubation with enzyme-treated chicory extract 3_3A (FIG. 7A). It is interesting to note that commercial preservative applied in standard 0.5 % concentration was able to inhibit the S. aureus growth only after 28 days. Non-treated chicory extract 3_3D putatively provided the bacteria with nutritional sources, such as carbohydrates, since the extract was not able to inhibit the growth during the one month follow-up. For P. aeruginosa, the effect of the 3_3A extract was similar than towards S. aureus, however the non-treated extract 3_3D was also effective after 7 days incubation (FIG. 7B). Interestingly, while commercial preservative was able to reduce the bacterial load in the beginning of the challenge test, the effect was reduced later, allowing the bacterial re-growth towards the end of the experimental time. 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