Microbiological and Physicochemical Evaluation of Hydroxypropyl Methylcellulose (HPMC) and Propolis Film Coatings for Cheese Preservation.

Dairy products are highly susceptible to contamination from microorganisms. This study aimed to evaluate the efficacy of hydroxypropyl methylcellulose (HPMC) and propolis film as protective coatings for cheese. For this, microbiological analyses were carried out over the cheese' ripening period, focusing on total mesophilic bacteria, yeasts and moulds, lactic acid bacteria, total coliforms, Escherichia coli, and Enterobacteriaceae. Physicochemical parameters (pH, water activity, colour, phenolic compounds content) were also evaluated. The statistical analysis (conducted using ANOVA and PERMANOVA) showed a significant interaction term between the HPMC film and propolis (factor 1) and storage days (factor 2) with regard to the dependent variables: microbiological and physicochemical parameters. A high level of microbial contamination was identified at the baseline. However, the propolis films were able to reduce the microbial count. Physicochemical parameters also varied with storage time, with no significant differences found for propolis-containing films. Overall, the addition of propolis to the film influenced the cheeses' colour and the quantification of phenolic compounds. Regarding phenolic compounds, their loss was verified during storage, and was more pronounced in films with a higher percentage of propolis. The study also showed that, of the three groups of phenolic compounds (hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids), hydroxycinnamic acids showed the most significant losses. Overall, this study reveals the potential of using HPMC/propolis films as a coating for cheese in terms of microbiological control and the preservation of physicochemical properties.

Antibacterial and Healing Effect of Chicha Gum Hydrogel (Sterculia striata) with Nerolidol.

Chicha gum is a natural polymer obtained from the Sterculia striata plant. The hydroxyl groups of its structure have a chemical affinity to form hydrogels, which favors the association with biologically active molecules, such as nerolidol. This association improves the biological properties and allows the material to be used in drug delivery systems. Chicha gum hydrogels associated with nerolidol were produced at two concentrations: 0.01 and 0.02 g mL-1. Then, the hydrogels were characterized by thermogravimetry (TG), Fourier Transform Infrared spectroscopy (FTIR), and rheological analysis. The antibacterial activity was tested against Staphylococcus aureus and Escherichia coli. The cytotoxicity was evaluated against Artemia salina. Finally, an in vivo healing assay was carried out. The infrared characterization indicated that interactions were formed during the gel reticulation. This implies the presence of nerolidol in the regions at 3100-3550 cm-1. The rheological properties changed with an increasing concentration of nerolidol, which resulted in less viscous materials. An antibacterial 83.6% growth inhibition effect was observed using the hydrogel with 0.02 g mL-1 nerolidol. The in vivo healing assay showed the practical activity of the hydrogels in the wound treatment, as the materials promoted efficient re-epithelialization. Therefore, it was concluded that the chicha hydrogels have the potential to be used as wound-healing products.

Comparative Methods to Evaluate the Antioxidant Capacity of Propolis: An Attempt to Explain the Differences.

Propolis is a natural product produced by bees that contains a complex mixture of compounds, including phenolic compounds and flavonoids. These compounds contribute to its biological activities, such as antioxidant capacity. This study analysed the pollen profile, total phenolic content (TPC), antioxidant properties, and phenolic compound profile of four propolis samples from Portugal. The total phenolic compounds in the samples were determined by six different techniques: four different Folin-Ciocalteu (F-C) methods, spectrophotometry (SPECT), and voltammetry (SWV). Of the six methods, SPECT allowed the highest quantification, while SWV achieved the lowest. The mean TPC values for these methods were 422 ± 98 and 47 ± 11 mg GAE/g sample, respectively. Antioxidant capacity was determined by four different methods: DPPH, FRAP, original ferrocyanide (OFec), and modified ferrocyanide (MFec). The MFec method gave the highest antioxidant capacity for all samples, followed by the DPPH method. The study also investigated the correlation between TPC and antioxidant capacity with the presence of hydroxybenzoic acid (HBA), hydroxycinnamic acid (HCA), and flavonoids (FLAV) in propolis samples. The results showed that the concentrations of specific compounds in propolis samples can significantly impact their antioxidant capacity and TPC quantification. Analysis of the profile of phenolic compounds by the UHPLC-DAD-ESI-MS technique identified chrysin, caffeic acid isoprenyl ester, pinocembrin, galangin, pinobanksin-3-O-acetate, and caffeic acid phenyl ester as the major compounds in the four propolis samples. In conclusion, this study shows the importance of the choice of method for determining TPC and antioxidant activity in samples and the contribution of HBA and HCA content to their quantification.

High Hydrostatic Pressure in the Modulation of Enzymatic and Organocatalysis and Life under Pressure: A Review.

The interest in high hydrostatic pressure (HHP) is mostly focused on the inactivation of deleterious enzymes, considering the quality-related issues associated with enzymes in foods. However, more recently, HHP has been increasingly studied for several biotechnological applications, including the possibility of carrying out enzyme-catalyzed reactions under high pressure. This review aims to comprehensively present and discuss the effects of HHP on the kinetic catalytic action of enzymes and the equilibrium of the reaction when enzymatic reactions take place under pressure. Each enzyme can respond differently to high pressure, mainly depending on the pressure range and temperature applied. In some cases, the enzymatic reaction remains significantly active at high pressure and temperature, while at ambient pressure it is already inactivated or possesses minor activity. Furthermore, the effect of temperature and pressure on the enzymatic activity indicated a faster decrease in activity when elevated pressure is applied. For most cases, the product concentration at equilibrium under pressure increased; however, in some cases, hydrolysis was preferred over synthesis when pressure increased. The compiled evidence of the effect of high pressure on enzymatic activity indicates that pressure is an effective reaction parameter and that its application for enzyme catalysis is promising.

Semi-Quantitative Discrimination of Honey Adulterated with Cane Sugar Solution by an ETongue.

This study successfully applied a potentiometric E-tongue with 20 cross-selectivity lipidic polymeric membranes in the discrimination of three semi-quantitative groups, that represented the following intervals of honey adulteration percentage with cane sugar: 0 %; [0, 10]%; [10, 20]% of adulteration. We analysed five different types of Portuguese honey; five brands of cane sugar were added to the adulterated samples; a comparative analysis was then performed. Linear discriminant analysis coupled with a tabu search algorithm for feature selection was applied to the ETongue's analytical data to select the best model. A discriminant model with 12 sensors was obtained. This model classified correctly all samples in both in internal (train data, 15 samples) and external validation (test data,10 samples). Also, multiple linear regression with tabu search was applied to verify if ETongue's data would allow quantifying the honey's adulteration level. The results showed that it was possible to obtain a quantitative model but with unsatisfactory predictive performance in the test data group (external validation), giving, in general, values below the expected concentrations. E-tongue is a real-time green, flexible and low-cost analytical tool that requires minimum sample preparation and no special technical skills, being a promising tool for everyday application.

High-Pressure-Based Strategies for the Inactivation of Bacillus subtilis Endospores in Honey.

Honey is a value-added product rich in several types of phenolic compounds, enzymes, and sugars recently explored in biomedical and food applications. Nevertheless, even though it has a low water activity (aW ≈ 0.65) that hinders the development of pathogenic and spoilage microorganisms, it is still prone to contamination by pathogenic microorganisms (vegetative and spores) and may constitute harm to special groups, particularly by immunosuppressed people and pregnant women. Thus, an efficient processing methodology needs to be followed to ensure microbial safety while avoiding 5-hydroxymethylfurfural (HMF) formation and browning reactions, with a consequent loss of biological value. In this paper, both thermal (pressure-assisted thermal processing, PATP) and nonthermal high-pressure processing (HPP), and another pressure-based methodology (hyperbaric storage, HS) were used to ascertain their potential to inactivate Bacillus subtilis endospores in honey and to study the influence of aW on the inactivation on this endospore. The results showed that PATP at 600 MPa/15 min/75 °C of diluted honey (52.9 °Brix) with increased aW (0.85 compared to ≈0.55, the usual honey aW) allowed for inactivating of at least 4.0 log units of B. subtilis spores (to below detection limits), while HS and HPP caused neither the germination nor inactivated spores (i.e., there was neither a loss of endospore resistance after heat shock nor endospore inactivation as a consequence of the storage methodology). PATP of undiluted honey even at harsh processing conditions (600 MPa/15 min/85 °C) did not impact the spore load. The results for diluted honey open the possibility of its decontamination by spores' inactivation for medical and pharmaceutical applications.

Role of Honey in Advanced Wound Care.

Honey is a natural product rich in several phenolic compounds, enzymes, and sugars with antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial potential. Indeed, the development of honey-based adhesives for wound care and other biomedical applications are topics being widely investigated over the years. Some of the advantages of the use of honey for wound-healing solutions are the acceleration of dermal repair and epithelialization, angiogenesis promotion, immune response promotion and the reduction in healing-related infections with pathogenic microorganisms. This paper reviews the main role of honey on the development of wound-healing-based applications, the main compounds responsible for the healing capacity, how the honey origin can influence the healing properties, also highlighting promising results in in vitro and in vivo trials. The challenges in the use of honey for wound healing are also covered and discussed. The delivery methodology (direct application, incorporated in fibrous membranes and hydrogels) is also presented and discussed.

Conventional and emergent technologies for honey processing: A perspective on microbiological safety, bioactivity, and quality.

Honey is a natural food of worldwide economic importance. Over the last decades, its potential for food, medical, cosmetical, and biotechnological applications has been widely explored. One of the major safety issues regarding such applications is its susceptibility to being contaminated with bacterial and fungi spores, including pathogenic ones, which may impose a hurdle to its consumption in a raw state. Another factor that makes this product particularly challenging relies on its high sugar content, which will lead to the formation of hydroxymethylfurfural (HMF) when heated (due to Maillard reactions). Moreover, honey's bioactivity is known to be affected when it goes through thermal processing due to its unstable and thermolabile components. Therefore, proper food processing methodologies are of utmost importance not only to ensure honey safety but also to provide a high-quality product with low content of HMF and preserved biological properties. As so, emerging food processing technologies have been employed to improve the safety and quality of raw honey, allowing, for example, to reduce/avoid the exposure time to high processing temperatures, with consequent impact on the formation of HMF. This review aims to gather the literature available regarding the use of conventional and emergent food processing technologies (both thermal and nonthermal food processing technologies) for honey decontamination, preservation/enhancement of honey biological activity, as well as the sensorial attributes.

Effect of extreme heat processing on the Moroccan Zantaz' honey antioxidant activities.

The effect of an extreme heat processing on Zantaz honey samples was studied using a panel of physicochemical parameters, antioxidant activities and FTIR-ATR spectroscopy. Honey samples were heated at 121 °C for 30 min and the heat processing effect was confirmed indirectly through the assessment of hydroxymethylfurfural content, for which the values increased significantly (p < 0.01), and diastase activity, which was totally absent after the thermal processing. Besides, the effects of the heat on the antioxidant activities were diverse. Indeed, while the ability to scavenge 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid cation radicals (ABTS·+) and 2,2-diphenyl-1-picrylhydrazyl radicals was enhanced (p < 0.05 only for ABTS·+), after the heat processing, nitric oxide radicals scavenging activity was reduced drastically (p < 0.01). Regarding the chelating power, it was totally abolished following heating. Other activities showed no significant alteration. The initial values of antioxidant activities seem to be determinant in the changes occurring after the heat processing. Amongst the analysed parameters, following the heat processing, the honey colour was the variable where the influence of baseline values was the greatest. The spectral analyses confirmed that FTIR-ATR is a useful technique to discriminate the chemical differences occurring in honey after heat processing.

Enzyme Inhibitory Potential of Ligustrum lucidum Aiton Berries.

Ligustrum lucidum Aiton and its berries have been used in Chinese traditional medicine for around two thousand years. In the present study, L. lucidium berries harvested in two regions of Portugal were studied. Haemolytic activity and inhibition of oxidative haemolysis as well as the enzyme inhibitory activities (α-amylase enzyme and acetylcholinesterase) were assessed. Results suggest that the different biological activities varied according to the region where samples were collected. Results demonstrated that the sample obtained from region R1 was the most efficient extract for all parameters evaluated, presenting the lowest values of IC50, 10.67 ± 0.46 µg/mL for the inhibition of erythrocyte oxidative haemolysis, 58.28 ± 3.77 µg/mL for the α-amylase enzyme and 67.67 ± 2.10 µg/mL for the acetylcholinesterase inhibition. L. Lucidum berries may be an interesting source of compounds for use in the development of the therapeutic armamentarium for diseases where enzymatic disruption is believed to play a role.

Kaempferol: A Key Emphasis to Its Anticancer Potential.

A marked decrease in human cancers, including breast cancer, bone cancer, and cervical cancer, has been linked to the consumption of vegetable and fruit, and the corresponding chemoprotective effect has been associated with the presence of several active molecules, such as kaempferol. Kaempferol is a major flavonoid aglycone found in many natural products, such as beans, bee pollen, broccoli, cabbage, capers, cauliflower, chia seeds, chives, cumin, moringa leaves, endive, fennel, and garlic. Kaempferol displays several pharmacological properties, among them antimicrobial, anti-inflammatory, antioxidant, antitumor, cardioprotective, neuroprotective, and antidiabetic activities, and is being applied in cancer chemotherapy. Specifically, kaempferol-rich food has been linked to a decrease in the risk of developing some types of cancers, including skin, liver, and colon. The mechanisms of action include apoptosis, cell cycle arrest at the G2/M phase, downregulation of epithelial-mesenchymal transition (EMT)-related markers, and phosphoinositide 3-kinase/protein kinase B signaling pathways. In this sense, this article reviews data from experimental studies that investigated the links between kaempferol and kaempferol-rich food intake and cancer prevention. Even though growing evidence supports the use of kaempferol for cancer prevention, further preclinical and clinical investigations using kaempferol or kaempferol-rich foods are of pivotal importance before any public health recommendation or formulation using kaempferol.

Microbiological Assessment, Nutritional Characterization and Phenolic Compounds of Bee Pollen from Mellipona mandacaia Smith, 1983.

This study aims to assess the microbiological parameters and the chemical composition of 21 samples of stingless bee pollen (Melipona mandacaia) from two regions of Bahia, Brazil (João Dourado and Uibaí), with particular emphasis on the nutritional value, total phenols and flavonoids and fatty acids composition. Regarding the microbiological quality, the studied microorganisms (moulds and yeasts, coliforms, Escherichia coli, Staphylococcus aureus, Salmonella sp., psychrotrophic and sulfite-reducing Clostridia) were absent in all samples. On the other hand, the values obtained for the aerobic mesophilic microorganism ranged from 11.0 ± 1.0 to 1.32 ± 1.2 cfu∙g-1 (JD samples) and from 282 ± 3.82 to 688 ± 10.1 cfu∙g-1 (U samples). The nutritional parameters (moisture, ash, water activity, pH, total acidity, protein, fiber, total phenolic, flavonoids and reducing sugars) were within the stipulated by law, except for pH and moisture content, which presented superior and inferior values, respectively. Polyunsaturated fatty acids (54.1%) were significantly higher than saturated (42.18%) and monounsaturated (3.71%). It was found that the bee pollen is safe from the microbiological point of view and has a good nutritional quality. The influence of the geographical origin on the assessed parameters was evident, especially concerning the fatty acid profile.

Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole.

BACKGROUND: The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered. RESULTS: MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation. CONCLUSIONS: PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C. glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C. glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C. glabrata and the associated worse prognosis of an infection by this species.

Transglutaminases: recent achievements and new sources.

Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, tissues, and body fluids, that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein or peptide-bound glutamine. Besides forming these bonds, that exhibit high resistance to proteolytic degradation, transglutaminases also form extensively cross-linked, generally insoluble, protein biopolymers that are indispensable for the organism to create barriers and stable structures. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have also been explored, but in a much lesser extent. Our group has identified a transglutaminase in the oomycete Phytophthora cinnamomi, which is able to induct defense responses and disease-like symptoms. In this mini-review, we report the achievements in this area in order to illustrate the importance and the versatility of transglutaminases.

Use of propolis in the sanitization of lettuce.

The present study aimed to determine the effectiveness of propolis in reducing the microbial load in ready-to-eat (RTE) and fresh whole head (FWH) lettuces (Lactuca sativa L.) type Batavia. Two sanitizing solutions were employed: sodium hypochlorite (SH) and propolis (PS), during 15 and 30 min. Tap water (TW) was used as a control. Regarding the mean reduction on aerobic mesophiles, psychrotrophic and fecal coliforms, the SH and PS treatments showed the same pattern of variation. In all cases, PS was slightly more effective in the microbiological reduction in comparison with commercial SH. Reductions between two and three log cycles were obtained with PS on aerobic mesophiles and psychrotrophic counts. The information obtained in the present study can be used to evaluate the potential use of propolis as product for sanitizing other vegetables and for developing other food preservation technologies, with impact on human health.

Developments in the fermentation process and quality improvement strategies for mead production.

Mead is a traditional alcoholic drink derived from the fermentation of diluted honey in the presence of appropriate yeast. Its modern production, in general terms, involves the addition of nutrients to initial diluted honey, pasteurization, yeast inoculation, fermentation and removal of impurities. Undesirable events along the process have been reported; among them, we highlight: delayed or arrested fermentations, modified and unpleasant sensory and quality parameters of the final product. These problems have been linked to the inability of yeasts to accomplish their role in extreme growth conditions. Emphasis has also been placed on the long fermentation times required, ranging from weeks to months, particularly when traditional procedures are applied and when the honey concentration is low. A series of alterations to the must and technological changes have been proposed in order to optimize the mead production process. In this context, this review examines the evidence that aims to improve meads' quality and make the production process easier and more efficient, by clarifying the source of unexpected events, describing the implementation of different fermentative microorganisms and using new methodologies.

The High Pressure Preservation of Honey: A Comparative Study on Quality Changes during Storage.

In commercially available honey, the application of a heat treatment to prevent spoilage can potentially compromise its beneficial properties and quality, and these effects worsen with extended storage. The high-pressure processing (HPP) of honey is being explored, but its long-term impact on honey quality has not been characterised yet. This study evaluated the effects of HPP and thermal processing on the microbial load, physicochemical quality (i.e., hydroxymethylfurfural content and diastase activity), and antioxidant capacity of honey after treatment and following extended storage (6, 12, and 24 months) at 20 °C. Pasteurization (78 °C/6 min) effectively eliminated the microorganisms in honey but compromised its physicochemical quality and antioxidant activity. HPP initially showed sublethal inactivation, but storage accelerated the decrease in yeasts/moulds and aerobic mesophiles in honey (being <1 log CFU/g after 24 months of storage) compared to unprocessed honey and honey thermally treated under mild conditions (55 °C/15 min). The physicochemical characteristics of the quality of HPP-treated honey and raw unprocessed honey did change after long-term storage (24 months) but remained within regulatory standards. In conclusion, HPP emerged as a more suitable and safe preservation method for Apis mellifera honey, with a minimal risk of a loss of antioxidant activity compared to traditional industrial honey pasteurization.

Fermented Grapevine Leaves: Potential Preserving Agent in Yogurt.

In this study, we monitored the fermentative process of Vitis vinifera L. leaves (grapevine), spontaneously or promoted by Saccharomyces cerevisiae, in both solid and liquid media. We also aimed to evaluate the effect on the bioactivity and shelf life of yogurt incorporating fermented and non-fermented grapevine leaves compared to yogurt produced with the preservative potassium sorbate. The results revealed that fermented grapevine leaf extracts increased their bioactive compounds and antioxidant activity, particularly in fermentations in a solid medium. In yogurt samples with incorporation extract from solid spontaneous fermentation and extract from solid yeast fermentation, even in small quantities, they exhibited higher levels of total phenols (1.94 and 2.16 mg GAE/g of yogurt, respectively) and antioxidant activity (5.30 and 5.77 mg TroloxE/g of yogurt; and 1.33 and 1.34 mg Fe(II)E/g of yogurt, respectively) compared to control yogurt (1.44 mg GAE/g of yogurt, 4.00 mg TroloxE/g of yogurt, and 1.01 mg Fe(II)E/g of yogurt). Additionally, yogurts supplemented with fermented grapevine leaves demonstrated the potential to inhibit microbial growth without impairing the multiplication of lactic acid bacteria.

Plant-Based Beverages: Consumption Habits, Perception and Knowledge on a Sample of Portuguese Citizens.

Plant-based beverages (PBB) consumption has increased significantly worldwide due to an interest in vegetarian/vegan diets, taste preferences, health and ethical and environmental issues. Therefore, this study intends to investigate consumption habits, consumer preferences and consumers' level of knowledge about PBB. In this study, a voluntary, anonymous questionnaire survey was applied to a sample of participants from Portugal. The sample was recruited by convenience, and therefore, the distribution among the groups was not even. Data analysis involved different statistical techniques: basic statistics, chi-square tests, factor analysis, cluster analysis and tree classification analysis. The results indicated that the most consumed PBB were almond, soy and oat beverages. The majority of consumers chose these beverages for nutritional and health reasons, while a smaller number consumed them as part of a vegetarian or vegan diet. The main motivations for consuming PBB are mainly associated with sustainability and health benefits. The results regarding the respondents' knowledge about PBB revealed that a health-related profession was the most significant predictor. These results allowed us to conclude that the factors of nutrition, health, ethics and practice of a vegetarian/vegan diet influence the consumption of PBB. It was also concluded that being in a healthcare profession, along with age and professional status within this field, were significant factors influencing the level of knowledge about PBB.