Plant-Based Alternatives to Mold-Ripened Cheeses as an Innovation among Dairy Analogues.

There is a growing demand for vegan products and plant-based food when dealing with the impact of livestock on the climate crisis. The aim of this study was to develop a formulation for a plant-based analogue of mold-ripened cheese. Were investigated the following plant materials: cashews, pistachios, soy flour, chickpea flour, pea protein, pumpkin protein, hemp protein, and spirulina powder. Plant matrices were fermented with lactic acid bacteria (LAB) starter cultures and cheese starter cultures of mold species Geotrichum candidum and Penicillium camemberti. All microorganisms' growth were tested in a vegan-type culture medium. Calcium supplementation was applied and followed by an in-depth analysis of the elemental composition of selected analogues with inductively coupled plasma optical emission spectroscopy. The physicochemical and organoleptic analyses of plant-based alternatives of Camembert were conducted. This is the first paper describing novel formulations for plant-based alternatives for Camembert cheese prepared with techniques mimicking the original milk product.

Do they make a good match? Molecular dynamics studies on CALB-catalyzed esterification of 3-phenylpropionic and cinnamic acids.

Lipases are versatile catalysts widely used in industrial biotransformations and laboratory-scale developed reactions with industrial potential. Despite the fact that lipase B from Candida antarctica (CALB) is one of the most widely used lipolytic enzymes, its substrate specificity is still poorly understood. One observed trend is that reactions carried out with carboxylic acids containing a double bond are less efficient on average. Here, we have utilized a combination of in vitro and in silico techniques, to better understand the negative impact of a double bond on CALB-mediated esterification. Then through extensive molecular dynamics (MD) simulations, we were able to map the entry pathway of cinnamic acid and its derivative into the CALB active site, and their interactions with catalytic residues. We observed a 2 step binding mechanism of studied compounds, where they first penetrate the enzyme pocket in a conformation where their carboxylic groups are extended towards the solvent. This is followed by further penetration of the acid into the enzymatic active pocket, and a full rotation within the active site, which orients the acid in a conformation that allows further steps of the esterification reaction. As acids containing a double bond are more rigid, their mobility and thus ability to rotate in the narrow CALB active site is hampered, which provides a structural explanation for the decreased efficiency of such acids. Our data provide insight into the substrate specificity of CALB-mediated esterification, providing important structural guidelines to better understand and potentially improve the efficiency of such reactions.

The Effect of Plasma-Treated Water on Microbial Growth and Biosynthesis of Gamma-Decalactones by Yarrowia lipolytica Yeast.

In recent years, the production of plasma-treated water (PTW) by low-temperature low-pressure glow plasma (LPGP) has been increasingly gaining in popularity. LPGP-treated water changes its physical and physiochemical properties compared to standard distilled water. In this study, a non-conventional lipolytic yeast species Yarrowia lipolytica was cultivated in culture media based on Nantes plasma water with heightened singlet oxygen content (Nantes PW) or in water treated with low-temperature, low-pressure glow plasma while in contact with air (PWTA) or nitrogen (PWTN). The research aimed to assess the influence of culture conditions on castor oil biotransformation to gamma-decalactone (GDL) and other secondary metabolites in media based on nanowater. The Nantes plasma water-based medium attained the highest concentration of gamma-decalactone (4.81 ± 0.51 g/L at 144 h of culture), maximum biomass concentration and biomass yield from the substrate. The amplified activity of lipases in the nanowater-based medium, in comparison to the control medium, is encouraging from the perspective of GDL biosynthesis, relying on the biotransformation of ricinoleic acid, which is the primary component of castor oil. Although lipid hydrolysis was enhanced, this step seemed not crucial for GDL concentration. Interestingly, the study validates the significance of oxygen in ß-oxidation enzymes and its role in the bioconversion of ricinoleic acid to GDL and other lactones. Specifically, media with higher oxygen content (WPTA) and Nantes plasma water resulted in remarkably high concentrations of four lactones: gamma-decalactone, 3-hydroxy-gamma-decalactone, dec-2-en-4-olide and dec-3-en-4-olide.

Food Toxicology and Food Safety: Report of the 3rd International Electronic Conference on Foods: Food, Microbiome, and Health-A Celebration of the 10th Anniversary of Foods' Impact on Our Wellbeing.

The purpose of the conference session summarized in this article was to bring together international experts on food toxicology and food safety and share the current scientific knowledge on these topics. The presentations covered a wide range of interdisciplinary issues, including (i) the impact of diet on body weight and health outcomes including results from animal models of carcinogenesis, (ii) methods for microbial oil extraction, (iii) food processing and its impact on food safety and health, (iv) novel compounds to avoid mycotoxin contamination of agricultural products, and (v) the safety of cannabidiol in food supplements based on Cannabis sativa extracts. Some of the conclusions of the presentations included that correct food choices may impact on the risk of non-communicable diseases such as cancer, that food processing may have an influence on health, by either reducing or increasing risks, and that research regarding novel compounds is important, which may have preventive but also detrimental effects on health.

Lipase-Catalyzed Synthesis, Antioxidant Activity, Antimicrobial Properties and Molecular Docking Studies of Butyl Dihydrocaffeate.

Green chemistry approaches, such as lipase-catalyzed esterification, are promising methods for obtaining valuable chemical compounds. In the case of the use of lipases, unlike in aqueous environments, the processes of the ester bond formations are encountered in organic solvents. The aim of the current research was to carry out the lipase-catalyzed synthesis of an ester of dihydrocaffeic acid. The synthesized compound was then evaluated for antioxidant and antimicrobial activities. However, the vast majority of its antioxidant activity was retained, which was demonstrated by means of DPPH· (2,2-diphenyl-1-picrylhydrazyl) and CUPRAC (cupric ion reducing antioxidant capacity) methods. Regarding its antimicrobial properties, the antifungal activity against Rhizopus oryzae is worth mentioning. The minimum inhibitory and fungicidal concentrations were 1 and 2 mM, respectively. The high antifungal activity prompted the use of molecular docking studies to verify potential protein targets for butyl ester of dihydrocaffeic ester. In the case of one fungal protein, namely 14-α sterol demethylase B, it was observed that the ester had comparable binding energy to the triazole medication, isavuconazole, but the interacted amino acid residues were different.

Mini-Review on the Enzymatic Lipophilization of Phenolics Present in Plant Extracts with the Special Emphasis on Anthocyanins.

Different plant extracts have the potential to be important sources of phenolic compounds. Their antibacterial, antifungal and antioxidant properties are of interest to researchers due to various possibilities for use in the pharmacy, cosmetic and food industries. Unfortunately, the direct application of phenolics in food is limited because of their hydrophilic nature and low solubility. The review is devoted to the recent advances in the methods of lipophilization of phenolic extracts along with the use of enzymes. The concept of extract modification instead of single compound modification is based on the expected synergistic effect of many phenolic compounds. The main focus is on the phenolic compounds found in fruits, flowers and leaves of different common and underutilized as well as medicinal, folk-medicinal or endemic plants. The compiled papers point to the great interest in the modification of anthocyanins, highly active but often unstable phenolics. Some examples of other flavonoids are also outlined. The possible applications of the lipophilized plant extracts are presented for improving the stability of edible oils, decreasing the content of acrylamide, exhibiting higher color stability in thermal processing and increasing the nutritional value.

Expression Profile of Selected Genes Involved in Storage Lipid Synthesis in a Model Oleaginous Yeast Species Yarrowia lipolytica.

Yarrowia lipolytica yeast is a model species of the group of oleaginous microorganisms capable of intracellular lipids accumulation in an amount exceeding 20% of the dry mass. Single cell oil biosynthesis can follow one of two biochemical pathways-de novo accumulation of cellular lipids in medium containing non-lipid carbon sources (including saccharides, glycerol) and ex novo microbial oil synthesis which involves fatty acids uptake from the environment. The mRNA expression of selected genes of de novo and ex novo lipid synthesis pathways was analyzed and correlated with the phenotypically observed features. It was proved that the accumulation yield of storage lipids via ex novo pathway was to some extent dependent on the limitation of the nitrogen source in the medium. It was also proposed that the synthesis of intracellular lipids in lipid-rich medium proceeded mainly via ex novo pathway, although the activity of genes encoding the enzymes of the de novo pathway were not completely inhibited at the stage of transcription by fatty acids present in the medium (e.g., ATP-citrate lyase). Molecular markers of two biosynthesis routes has been outlined and a hypothetical connection point between de novo and ex novo route were indicated.

Recent advances in the enzymatic synthesis of lipophilic antioxidant and antimicrobial compounds.

Due to the increase in the consumption of highly processed food in developed countries, as well as, a growing number of foodborne diseases, exploration of new food additives is an issue focusing on scientific attention and industrial interest. Functional compounds with lipophilic properties are remarkably desirable due to the high susceptibility to the deterioration of lipid-rich food products. This paper in a comprehensive manner provides the current knowledge about the enzymatic synthesis of lipophilic components that could act as multifunctional food additives. The main goal of enzymatic lipophilization of compounds intentionally added to food is to make these substances soluble in lipids and/or to obtain environmentally friendly surfactants. Moreover, lipase-catalyzed syntheses could result in changes in the antioxidant and antimicrobial activities of phenolic compounds, carbohydrates, amino acids (oligopeptides), and carboxylic acids. The review describes also the implementation of a new trend in green chemistry, where apart from simple and uncomplicated chemical compounds, the modifications of multi-compound mixtures, such as phenolic extracts or essential oils have been carried out.

Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis.

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2-6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

Enzymatic Synthesis of Lipophilic Esters of Phenolic Compounds, Evaluation of Their Antioxidant Activity and Effect on the Oxidative Stability of Selected Oils.

The aim of the study was to compare the effect of the substituent and its position in the aromatic ring on the antioxidant activity of hexanoic acid esters obtained in reactions catalyzed by immobilized lipase B from Candida antarctica. 4-Hydroxybenzyl hexanoate, 2-hydroxybenzyl hexanoate, 4-methoxybenzyl hexanoate, and vanillyl hexanoate were obtained with conversion yields of 50 to 80%. The antioxidant activity of synthesized esters, their alcohol precursors and BHT (Butylated HydroxyToluene) was compared with DPPH (2,2-diphenyl-1-picrylhydrazyl), CUPRAC (cupric ion reducing antioxidant capacity), and CBA (crocin bleaching assay) methods. Furthermore, it was investigated whether the presence of vanillyl hexanoate in a concentration of 0.01 and 0.1% affected the oxidative stability of sunflower and rapeseed oils in the Rancimat test. It was observed that the antioxidant activity of hexanoic acid esters depends on the presence and position of the hydroxyl group in the aromatic ring. The highest activities were found for vanillyl alcohol, vanillyl hexanoate, and BHT. The addition of the ester and BHT significantly extended the induction times of the tested oils, and these compounds exhibited similar activity. Vanillyl hexanoate increased the induction time from 4.49 to 5.28 h and from 2.73 to 3.12 h in the case of rapeseed and sunflower oils, respectively.

Studies on Upgradation of Waste Fish Oil to Lipid-Rich Yeast Biomass in Yarrowia lipolytica Batch Cultures.

The aim of the study was to evaluate the possibility to utilize a fish waste oil issued from the industrial smoking process in nitrogen-limited Yarrowia lipolytica yeast batch cultures. The waste carbon source was utilized by the yeast and stimulated the single cell oil production via an ex novo pathway. The yeast biomass contained lipids up to 0.227 g/g d.m.. Independently from culture conditions, high contents of very long chain fatty acids were quantified in yeast biomass including docosahexaenoic (DHA), eicosapentaenoic acid (EPA), eicosenic and erucic acids. The pH regulation did not influence the cellular lipids yield (0.234 g/g d.m.). Meanwhile, the intensification of the oxygenation of medium by changing the mixing speed (maximum concentration of lipids produced 4.64 g/dm3) and decreasing the amount of inoculum had a positive effect on the culture parameters in waste fish oil medium. Further work on upgradation of the original waste is advisable, especially because the oil indicated high content of polyphenols and lower susceptibility to oxidation than microbial oil derived from control olive oil medium.

Application of freeze-dried Yarrowia lipolytica biomass in the synthesis of lipophilic antioxidants.

OBJECTIVE: The aim of the study was to evaluate the possibility of using Y. lipolytica biomass as a whole-cell catalyst in the synthesis of lipophilic antioxidants, with the example of esterification of five phenolic acids with 1-butanol. RESULTS: Freeze-dried Y. lipolytica biomass was successfully applied as a biocatalyst in the synthesis of esters of phenylpropanoic acid derivatives with 75-98% conversion. However, in the case of phenylacetic acid derivatives, results below 10% were obtained. The biological activity of phenolic acid esters was strongly associated with their chemical structures. Butyl 3-(4-hydroxyphenyl)propanoate showed an IC50 value of 19 mg/ml (95 mM) and TEAC value of 0.427. Among the compounds tested, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited the highest antifungal activity. CONCLUSIONS: Lipophilization of phenolic acids achieved by enzymatic esterification creates prospects for using these compounds as food additives with antioxidant properties in lipid-rich food matrices.

Effect of Electron-Beam Radiation and Other Sterilization Techniques on Structural, Mechanical and Microbiological Properties of Thermoplastic Starch Blend.

This work investigates the potential application of various sterilization methods for microorganism inactivation on the thermoplastic starch blend surface. The influence of the e-beam and UV radiation, ethanol, isopropanol and microwave autoclave on structural and packaging properties were studied. All the applied methods were successful in the inactivation of yeast and molds, however only the e-beam radiation was able to remove the bacterial microflora. The FTIR analysis revealed no significant changes in the polymer structure, nevertheless, a deterioration of the mechanical properties of the blend was observed. The least invasive method was the UV radiation which did not affect the mechanical parameters and additionally improved the barrier properties of the tested material. Moreover, it was proved that during the e-beam radiation the chain scission and cross-linking occurred. The non-irradiated and irradiated samples were subjected to the enzymatic degradation studies performed in the presence of amylase. The results indicated that irradiation accelerated the decomposition of material, which was confirmed by the measurements of weight loss, and mass of glucose and starch released to the solution in the course of biodegradation, as well as the FTIR and thermal analysis.

Screening of solvents for favoring hydrolytic activity of Candida antarctica Lipase B.

Lipases are a group of enzymes of considerable significance in organic synthesis, among which Candida antarctica lipase B (CALB) is one of the most widely studied enzymes. The activity of the biocatalyst has been intensively characterized in many organic media, but this paper aimed to compare the effect of 20 different solvents on the activity of CALB in the hydrolysis of p-nitrophenyl laurate. Nonpolar, polar aprotic, and polar protic solvents were used for enzyme pretreatment and then entered the composition of mixed solvents reaction medium. An impact of solvents on solvation processes affecting the catalysis steps, protein denaturation, and changes of its conformation was discussed. Moreover the hydrolytic activity of CALB with partition coefficient (logP) of the solvent used was correlated. It was emphasized that the substrate solubility plays an important role in solvent selection. In the presence of hydrophobic solvents, hydration layer becomes more hydrophobic facilitating the substrate access to the enzyme surface. In turn, polar compounds are good solvents for organic substrates facilitating the penetration of the aqueous layer that surrounds the surface of the enzyme. Two variants proved to be favorable for ester hydrolysis reaction: isooctane or polar solvent such as acetone, tert -butyl methyl ether, tert-butanol or acetonitrile.

An Insight into Storage Lipid Synthesis by Yarrowia lipolytica Yeast Relating to Lipid and Sugar Substrates Metabolism.

Single cell oil (SCO) is the lipid accumulated in the cells of oleaginous microorganisms. Cellular lipids can be synthesized in two different pathways: de novo by metabolizing hydrophilic substrates and ex novo by fermenting hydrophobic substrates. The aim of the study was to evaluate the effect of carbon source (glucose and olive oil) in the culture medium on the course of microbial oil accumulation in Y.lipolytica cells. The level of selected gene expression by real time quantitative PCR method was investigated. The significant increase in expression of the POX2 gene encoding acyl-CoA oxidase II, which preferentially oxidizes long-chain acyl-CoAs formed from substrate fatty acids incorporated inside the microbial cell, was observed in medium with olive oil in relation to glucose containing medium. Noteworthily, the presence of lipid carbon substrate did not inhibit the level of ACL gene transcription coding for ATP-citrate lyase, the key enzyme of the lipid de novo accumulation process. The present study indicated that de novo lipid biosynthesis could occur despite the presence of fatty acids in the medium, and the synthesis of storage lipids in the presence of lipid carbon substrates could be carried out with the use of both pathways (de novo and ex novo).

Trends in designing microbial silage quality by biotechnological methods using lactic acid bacteria inoculants: a minireview.

Ensiling is one of the best known method to preserve fodder. The forage before ensiling intended for silages usually contains a low number of lactic acid bacteria (LAB), so it is necessary to apply starter cultures of selected strains. Traditionally, LAB starter cultures were applied to lower the pH by producing lactic acid and to inhibit the growth of undesirable epiphytic microorganisms by competing for nutrients. Nowadays, LAB inoculants have become an effective tool for creating microbial quality of silages by selecting species with extraordinary features. Epiphytic microflora characteristic of plant material used for the production of silages and the sources of undesirable microflora in the ensiling process are discussed. This review focuses on the most frequently studied issues related to the microbial silage quality and the recent trends in increasing the quality by LAB inoculants, with respect to recent directions for selecting types of modern LAB for inoculation. Among them, the main trends described were prevention of the growth of filamentous fungi and detoxification of mycotoxins by LAB inoculants, inhibition of yeast growth by LAB present in preparations and limiting the development of pathogenic bacterial microflora through controlled fermentation with the participation of LAB and the presence of their metabolites.

Yarrowia lipolytica: a beneficious yeast in biotechnology as a rare opportunistic fungal pathogen: a minireview.

Yarrowia lipolytica is one of the most studied "non-conventional" yeast species capable of synthesizing a wide group of valuable metabolites, in particular lipases and other hydrolytic enzymes, microbial oil, citric acid, erythritol and γ-decalactone. Processes based on the yeast have GRAS status ("generally recognized as safe") given by Food and Drug Administration. The majority of research communications regarding to Y. lipolytica claim that the yeast species is non-pathogenic. In spite of that, Y. lipolytica, like other fungal species, can cause infections in immunocompromised and critically ill patients. The yeast possess features that facilitate invasion of the host cell (particularly production of hydrolytic enzymes), as well as the protection of the own cells, such as biofilm formation. The aim of this study was to present well-known yeast species Y. lipolytica as a rare opportunistic fungal pathogen. Possible pathogenicity and epidemiology of this yeast species were discussed. Antifungal drugs susceptibility and increasing resistance to azoles in Y. lipolytica yeasts were also presented.

Phenolic Acids Derivatives - Biotechnological Methods of Synthesis and Bioactivity.

Phenolic acids are secondary plant metabolites belonging to polyphenol classes, widely spread throughout the plant kingdom. The name "phenolic acids", in general, describes phenols that possess one carboxylic acid functionality and they could be divided into three major subclasses: hydroxybenzoic, hydroxycinnamic and hydroxyphenylacetic acids. The great interest in phenolic acids is associated with their high potential for food preservation (antioxidant and antimicrobial activity) and, last but not least, high therapeutic potential. The aim of this review is to summarise the current knowledge concerning phenolic acids and the topics discussed include natural sources of phenolic acids, biosynthesis and metabolism, health benefits of phenolic acids (first of all the antioxidant activity of dietary phenolics and also anticarcinogenic and anti-inflammatory effect), their antimicrobial activity for selected groups of bacteria and fungi (yeasts) and structural modifications of the molecule especially those increasing lipophilicity and improving solubility in lipids. Recent advances in the methods of enzymatic synthesis of phenolic acid derivatives are described and some conclusions referring to Structure-Property-Activity Relationships of phenolic acids, particularly important from the point of view of their pharmaceutical and nutritional applications, are discussed.

Synthetic and Natural Lipase Inhibitors.

Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including ß-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are ß- lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries and lipases are essential enzymes for lipid absorption - thus fat absorption or obesity can be controlled by lipase inhibition, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

Evaluation of the ability to metabolize 1, 2-propanediol by heterofermentative bacteria of the genus Lactobacillus

Background: New directions of research on lactic acid bacteria include investigation of metabolic pathways for the synthesis and/or metabolism of 1,2-propanediol, commonly used in the food and chemical industry, medicine, pharmacy and cosmetology as well as agriculture. The objective of this study was to compare the capacity of strains representing three diverse heterofermentative species belonging to the genus Lactobacillus to synthesize and/or transform 1,2-PD as well as to suggest new directions of research aimed at commercial use of this metabolite. Results: The novel strain of Lactobacillus buchneri A KKP 2047p, characterized as exhibiting an unusual trait for that species in the form of capacity to metabolize 1,2-PD, grew poorly in a medium containing 1,2-PD as a sole carbon source. The supplementation with glucose facilitated rapid growth of bacteria and use of 1,2-PD for the synthesis of propionic acid. A similar observation was noted for Lactobacillus reuteri. On the other hand, Lactobacillus diolivorans effectively metabolized 1,2-PD which was the sole carbon source in the medium, and the addition of glucose inhibited the synthesis of propionic acid. The experiments also investigated the effect of cobalamin as a diol dehydratase coenzyme involved in the propionic acid synthesis from 1,2-PD whose addition promoted the yield of the reaction in the case of all tested strains. Conclusions: All tested isolates showed the ability to effectively metabolize 1,2-PD (in the presence of cobalamin) and its conversion to propionic acid, which reveals that investigated bacteria meet the essential requirements of microorganisms with a potential application.