Microbial lipases and their application

Diplomová práce je zaměřena na studium preparátu do odlučovačů tuků a odpadních potrubí, který obsahuje mikroorganismy s lipolytickou aktivitou. Teoretická část práce popisuje lipázy, mikroorganismy, které tyto enzymy produkují a využití lipáz. V této části jsou také uvedeny možnosti identifikace mikroorganismů. Praktická část se zabývá studiem komerčního přípravku Sany Duo Spezial s prokázanou přítomností mikroorganismů s lipolytickou aktivitou. U těchto mikroorganismů byla provedena identifikace metodou PCR. Pomocí této metody byly mikroorganismy identifikovány jako rod Bacillus sp. Další charakteristika přípravku byla zaměřena na stanovení hodnoty CHSKCr a výzkum vlivu různých podmínek kultivačního prostředí na produkci lipáz a jejich aktivitu. Studovaný byl vliv teploty, iontů a pH. Lipolytická aktivita byla stanovována spektrofotometricky s využitím činidla p-nitrofenyllaurátu, který se štěpí na žlutý produkt p-nitrofenol.This diploma thesis is focused on the study of the preparation for fat separators and wastewater pipes that contains the microorganisms with lipolytic activity. Theoretical part of this thesis describes lipases, microorganisms producing this enzymes and usage of lipases. In this part possibilities of identification of microorganisms are presented too. The practical part is concerned with the study of commercial preparation Sany Duo Spezial with proven presence of microorganisms with lipolytic activity. These microorganisms were identified by means of the PCR method. This method identified mictoorganisms like genus Bacillus sp. Next characterization of the preparation was focused on the determination of COD and the investigation of the influence of various conditions of culture medium on the lipases production and their activity. The effect of temperature, ions and pH was studied. Lipolytic activity was determine spectrophotometricaly with usage of p-nitrophenyllaurate whitch dissociates to yellow product p-nitrophenol.

Microorganisms with lipoplytic activity and their applications

Lipasy jsou hydrolytické enzymy produkované řadou mikroorganismů. Tato práce popisuje nejen samotné mikroorganismy, které lipasy produkují, ale také různé možnosti průmyslového využití lipas. Lipasy nacházejí široké uplatnění v reakcích, ve kterých je nutná kombinace lipofilního substrátu s hydrofilním – při syntézách mastných esterů kyseliny askorbové nebo esterů cukru, při syntézách lipoaminokyselin nebo při lypolýze derivátů fenolu. Význam mají také vzhledem k ochraně životního prostředí, např. při čištění odpadních vod. V této práci byla lipolytická aktivita testována na pěti různých komerčních preparátech obsahujících mikroorganismy s lipolytickou aktivitou. Na stanovení lipolitické aktivity existuje řada různých metod. Příkladem může být spektrofotometrické stanovení lipolytické aktivity, při kterém se využívá schopnosti lipas štěpit p-nitrofenyllaurát na barevný produkt p-nitrofenol. Přičemž p-nitrofenol je detekován spektrofotometricky.Lipases are hydrolytic enzymes that are produced by many types of microorganisms. This thesis describes not only the microorganisms which produce lipases but also different possibilities for the industrial utilization of lipases. Lipases are widely used in reactions where the combination of a lipophilic substrate with a hydrophilic one is necessary – in the synthesis of ascorbic acid fatty esters, sugar esters, lipoaminoacids and in the lipophilization of phenolic derivatives. Lipases are also important in relation to environmental protection. For example, they are used for the purification of waste water. In this thesis, five different preparations containing microorganisms with lipolytic activity were tested for lipolytic activity. There are many ways of determining lipolytic activity. The spectrophotometric determination of lipolytic activity that uses the ability of lipases to divide p-nitrophenyllaurate to p-nitrophenol may serve as an example. After this p-nitophenol is detected spectrophotometricaly.

Biotechnological preparation of gelatines from chicken feet

In the European Union (EU), about five tons of poultry by-product tissues are produced every year. Due to their high collagen content, they represent a significant raw material source for gelatine production. The aim of the paper was the biotechnological preparation of gelatine from chicken feet. The influence of selected process factors on the gelatine yield, gel strength, viscosity, and ash of gelatine was observed; a two-level factor design of experiments with three variable process factors (enzyme addition, enzyme treatment time, and gelatine extraction time) was applied. After grinding and separating soluble proteins and fat, the purified raw material was treated in water at pH 7.5 with the addition of endoprotease at 23 degrees C and after thorough washing with water at 80 degrees C, gelatine was extracted. By the suitable choice of process conditions, gelatine with high gel strength (220-320 bloom), low ash content (s can be prepared. The extraction efficiency was 18-38%. The presented technology is innovative mainly by the enzymatic processing of the source raw material, which is economically, technologically, and environmentally beneficial for manufacturers. Chicken gelatines are a suitable alternative to gelatines made from mammals or fish, and can be used in many food, pharmaceutical, and biomedical applications.Internal Grant Agency of the Faculty of Technology, Tomas Bata University in Zlin [IGA/FT/2019/003

Hydration and barrier properties of emulsions with the addition of keratin hydrolysate

Although keratin hydrolysates (KH) are added to skin care agents, detailed studies on the moisturising effects of KH are lacking. The aim of this study is to test whether adding KH into an ointment base (OB) heighten hydration of the skin and diminish transepidermal loss of water (TEWL). Formulations containing 2%, 4%, and 6% of KH (based on OB weight) were prepared. Hydration, TEWL and skin pH were measured; intervals of measurements were as follows: 1, 2, 3, 4, 24 and 48 h. Testing was carried out on 10 men. In terms of hydration, supplementing the OB with 2% KH is optimal, as an 11-19% increase occurs in hydration of stratum corneum (SC). All the formulations with added KH as tested caused TEWL to decline after application. Keratin hydrolysate makes for an excellent occlusive; adding it to OB results in a 30-50% reduction in TEWL after application. KH functions as a humectant as well, as it helps to bind water from the lower layers of the epidermis to the SC. Formulations with additions of 2-6% of KH were stable in structure and did not cause phase separation even after 6 months storage. © 2018 by the authors

Stability and in vivo efficiency of natural cosmetic emulsion systems with the addition of vegetable oils

The aim of the paper is to test stability and biophysical properties of hydrophilic and lipophilic emulsions with selected vegetable seed oils: Limnanthes alba, Prunus amygdalus dulcis, Cannabis sativa, Rosa rubiginosa and Hellianthus annuus. Biophysical properties of emulsions are investigated in vivo using non-invasive instrumental methods (corneometry, tewametry and pH) in a group of 12 healthy women volunteers. Their stability profiles (colour, phase separation and centrifugation) under various temperatures (9, 25, 37 and 57 °C) and storage time (24 hours, 2, 7, 14, 21 and 28 days) were monitored. The moisturising activities of the emulsions supplemented with various oils were comparable. The lipophilic emulsions showed a better ability to improve the condition of the skin barrier due to formation of a surface lipid film. The tested formulations regulated the pH of the skin towards neutral values. Lipophilic emulsions showed earlier phase separation and changes in colour. The greatest resistance to thermal stress during storage was observed for the emulsion bases. Emulsions containing oils, except for those with rosehip and hempseed oils, were stable up to the temperature of 37 °C. The studied emulsion systems are excellent vehicles of vegetable oils and exhibit relatively good stability, benefiting the natural properties of skin

Preparation of keratin hydrolysate from chicken feathers and its application in cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs. © 2017 Journal of Visualized Experiments

Stability and in vivo efficiency of natural cosmetic emulsion systems with the addition of vegetable oils

The aim of the paper is to test stability and biophysical properties of hydrophilic and lipophilic emulsions with selected vegetable seed oils: limnanthes alba, Prunus amygdalus dulcis, Cannabis saliva, Rosa rubiginosa and Hellianthus annuus. Biophysical properties of emulsions are investigated in vivo using non-invasive instrumental methods (comeometry, tewametry and pH) in a group of 12 healthy women volunteers. Their stability profiles (colour, phase separation and centrifugation) under various temperatures (9, 25, 37 and 57 degrees C) and storage time (24 hours, 2, 7, 14, 21 and 28 days) were monitored. The moisturising activities of the emulsions supplemented with various oils were comparable. The lipophilic emulsions showed a better ability to improve the condition of the skin barrier due to formation of a surface lipid film. The tested formulations regulated the pH of the skin towards neutral values. Lipophilic emulsions showed earlier phase separation and changes in colour. The greatest resistance to thermal stress during storage was observed for the emulsion bases. Emulsions containing oils, except for those with rosehip and hempseed oils, were stable up to the temperature of 37 degrees C. The studied emulsion systems are excellent vehicles of vegetable oils and exhibit relatively good stability, benefiting the natural properties of skin

Enzyme conditioning of chicken collagen and taguchi design of experiments enhancing the yield and quality of prepared gelatins

During the production of mechanically deboned chicken meat (MDCM), a by-product is created that has no adequate use and is mostly disposed of in rendering plants. Due to the high content of collagen, it is a suitable raw material for the production of gelatin and hydrolysates. The purpose of the paper was to process the MDCM by-product into gelatin by 3-step extraction. An innovative method was used to prepare the starting raw material for gelatin extraction, demineralization in HCl, and conditioning with a proteolytic enzyme. A Taguchi design with two process factors (extraction temperature and extraction time) was used at three levels (42, 46, and 50 °C; 20, 40, and 60 min) to optimize the processing of the MDCM by-product into gelatins. The gel-forming and surface properties of the prepared gelatins were analyzed in detail. Depending on the processing conditions, gelatins are prepared with a gel strength of up to 390 Bloom, a viscosity of 0.9–6.8 mPa·s, a melting point of 29.9–38.4 °C, a gelling point of 14.9–17.6 °C, excellent water- and fat-holding capacity, and good foaming and emulsifying capacity and stability. The advantage of MDCM by-product processing technology is a very high degree of conversion (up to 77%) of the starting collagen raw material to gelatins and the preparation of 3 qualitatively different gelatin fractions suitable for a wide range of food, pharmaceutical, and cosmetic applications. Gelatins prepared from MDCM by-product can expand the offer of gelatins from other than beef and pork tissues. © 2023 by the authors

Hydration and barrier potential of cosmetic matrices with bee products

Honey, honey extracts, and bee products belong to traditionally used bioactive molecules in many areas. The aim of the study was primarily to evaluate the effect of cosmetic matrices containing honey and bee products on the skin. The study is complemented by a questionnaire survey on the knowledge and awareness of the effects and potential uses of bee products. The effect of bee molecules at various concentrations was observed by applying 12 formulations to the skin of the volar side of the forearm by non-invasive bioengineering methods on a set of 24 volunteers for 48 h. Very good moisturizing properties have been found in matrices with the glycerin extract of honey. Matrices containing forest honey had better moisturizing effects than those containing flower honey. Barrier properties were enhanced by gradual absorption, especially in formulations with both glycerin and aqueous honey extract. The observed organoleptic properties of the matrices assessed by sensory analysis through 12 evaluators did not show statistically significant differences except for color and spreadability. There are differences in the ability to hydrate the skin, reduce the loss of epidermal water, and affect the pH of the skin surface, including the organoleptic properties between honey and bee product matrices according to their type and concentration.Internal strategic project of Tomas Bata University in Zli

Effect of Oregano and Marjoram Essential Oils on the Physical and Antimicrobial Properties of Chitosan Based Systems

The effect of two essential oils (EOs), from Origanum vulgare and Origanum majorana, their structural properties, and concentration on physical and antimicrobial characteristics of chitosan based solutions as well as films was investigated. Results showed that significantly different behaviour was induced by variation in the compositions of given essential oils. Film-forming solutions (FFS) containing oregano oil comprised considerably greater particles in comparison with the marjoram samples. Similarly, structural changes were confirmed by SEM analysis of chitosan films modified with the EOs; the smaller particles of the marjoram oil demonstrated better compatibility with chitosan matrix. However, chitosan films enriched with the oregano oil showed significantly superior antimicrobial activity compared to the marjoram. The dissimilar effects of the two EOs were also observed by water vapour pressure (WVP) measurement; increasing the amount of oregano oil triggered a drop in the WVP of the prepared films, whereas the marjoram oil had a negligible impact in this respect. These results suggest that the structural features of active substances in the EOs play a crucial role in determining the final properties of FFS and biofilm systems. © 2017 Jana Sedlaříková et al