Comparison of Traditional and Industrial Sausages - Baranjski Kulen and Kulenova Seka Using Comprehensive Proteome, Peptidome and Metabolome Techniques

Research background. Baranjski kulen is one of the most popular fermented meat sausages originating from Croatia. It has protected geographical indication, and is traditionally produced in the Baranja region of Croatia. Kulenova seka is a fermented sausage very similar to Baranjski kulen, but it has a different caliber and consequently, a shorter time of production. In recent decades, due to the high demand and popularity of these products, industrially produced Baranjski kulen and Kulenova seka have become available on the market. This work aims to identify specific characteristics of traditional and industrial sausages, Baranjski kulen and Kulenova seka on proteome, peptidome and metabolome level which could potentially lead to better optimization of the industrial production process in order to obtain an equivalent to the traditional product. Experimental approach. Protein profiles of Baranjski kulen and Kulenova seka (traditional and industrial) were analysed using two-dimensional gel electrophoresis followed by differential display analysis and protein identification using mass spectrometry. Peptidomics profiling analysis was performed via liquid chromatography-tandem mass spectrometry. Furthermore, aroma profiles were investigated via headspace solid phase microextraction and gas chromatography-mass spectrometry. Results and conclusions. The major identified characteristics of each product were: industrial Baranjski kulen - specific degradation of MYH1 and TITIN, overabundance of stress-related proteins and increased phenylalanine degradation; traditional Baranjski kulen - decreased concentration of phenylalanine and overabundance of ALDOA and CAH3; industrial Kulenova seka - specific MYH4 and HBA degradation process; traditional Kulenova seka - overabundance of DPYD and MYL1, degradation of ALBU and MYG, decreased concentrations of almost all free amino acids and increased amounts of smoke derived volatile compounds. Novelty and scientific contribution. In this preliminary communication, the first insights into protein degradation processes and generation of peptides, free amino acids and aroma compounds of industrial and traditional Baranjski kulen and Kulenova seka are presented. Although further research is needed to draw general conclusions, the specific profile of proteins, peptides, amino acids, and volatile compounds represents the first step in the industrial production of sausages that meet the characteristics of traditional flavour.

Polyethylene Glycol-Stabilized Zein Nanoparticles Containing Gallic Acid

Research background. Gallic acid is a polyphenol presenting antioxidant and antitumor activities, however its use as a nutraceutical or drug is hindered by its low bioavailability. Zein is a natural protein found in corn and has been applied as nanoparticle for drug carrier. In this study, zein nanoparticles were obtained and stabilized with polyethylene glycol (PEG) as gallic acid carriers. Experimental approach. Nanoparticles were obtained by the liquid-liquid method and characterized in terms of mean size, polydispersity index, zeta potential, morphology, solid-state interactions, and encapsulation efficiency/drug loading. The stability of nanoparticles was evaluated in simulated gastrointestinal fluids and food simulants, and the antioxidant activity was determined by the scavenging of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Results and conclusions. Zein nanoparticles containing gallic acid were obtained and stabilized only in the presence of PEG. The optimal conditions originated nanoparticles with mean size <200 nm, low polydispersity index (<0.25) and negative zeta potential (20 mV). The gallic acid encapsulation efficiency was about 40 %, drug loading about 5 %, and the compound was encapsulated in an amorphous state. FTIR did not identify chemical interactions after gallic acid nanoencapsulation. Zein nanoparticles were more susceptible to release the gallic acid in gastric than intestinal simulated medium, however more than 50 % of drug content was protected from premature release. In food simulants, the gallic acid release from nanoparticles was prolonged and sustained. Moreover, the nanoencapsulation did not reduce the antioxidant activity of gallic acid. Novelty and scientific contribution. The results show the importance of PEG on the formation and properties of zein nanoparticles obtained by the liquid-liquid dispersion method. This study indicates PEG-stabilized zein nanoparticles are potential carriers for gallic acid delivery by the oral route to take advantage of its antioxidant properties and be applied both in the pharmaceutical and food industry.

Applicability of Saccharomyces cerevisiae Strains for the Production of Fruit Wines Using Cocoa Honey Complemented with Cocoa Pulp

Research background. Cocoa honey (CH) and cocoa pulp (CP) are both fruit pulps highly appreciated but, until now, CH is less processed than CP. In this work, it was investigated the applicability of strains of S. cerevisiae to ferment CH complemented with CP, to obtain fruit wines and improve CH commercialization. Experimental approach. The selection of a strain, previously isolated from cachaçaria distilleries in Brazil, took place based on its fermentation performance. The conditions for fermentation with S. cerevisiae L63 were then studied in relation to: volumetric proportion (φCH) of CH (complemented with CP), sucrose addition (γsuc), temperature (T) and inoculum size (No). The best conditions were applied in order to obtain fermentation profiles. Results and conclusions. S. cerevisiae L63 (No=107–108 cell/mL) is capable to ferment φCH of 90 and 80 % (V/V) for 24 or 48 h with γsuc of 50 and 100 g/L at T=28–30 °C resulting in wines with ethanol contents from 8 to 14 % (V/V). Additionally, the φCH=90 % (V/V) wine resulted in the lowest residual sugar concentration (<35 g/L) than the φCH=80 % (V/V) wine (~79 g/L) which could be classified as a sweet wine. In general, S. cerevisiae L63 resulted in a similar fermentation performance than a commercial strain tested, indicating its potential for fruit pulp fermentation. Novelty and scientific contribution. Therefore, S. cerevisiae L63 is capable to ferment CH complemented with CP to produce fruit wines with good commercial potentials that may also benefit small cocoa producers by presenting a product with greater added value.

Extraction of Lipophilic Antioxidants from Native Tomato Using Green Technologies

Research background. Tomato (Solanum lycopersicum L.) fruit is highly consumed worldwide and contains high levels of carotenoids and tocopherols, two powerful antioxidants. Native tomato genotypes are rarely used in large-scale commercialisation and industries but serve as a reservoir to diversify the species gene pool and can be employed to obtain functional compounds. Extraction methodologies are currently experimenting changes towards cleaner methodologies that are more efficient and environmentally friendly, including avoiding toxic or polluting solvents. Experimental approach. In this study, factorial and fractional factorial designs were used to evaluate the efficiency of digestive enzymes, sonication and green solvents to extract carotenoids and tocopherols from native tomatoes. Results and conclusions. Digestive enzymes and sonication increased the carotenoid content and the antioxidant activity of the obtained extracts when applied individually. However, when these treatments were applied together and in combination with green solvents (ethyl lactate and isopropyl acetate), the obtained extracts had the highest carotenoid and tocopherol contents as well as the maximal antioxidant activity, compared with non-combined treatments. Moreover, a correlation analysis suggested that antioxidant activity resulted from synergistic effects between several antioxidants rather than individual compounds. The tomato extracts were obtained through a relatively rapid extraction method (2 h) and maintained their functional activity. Novelty and scientific contribution. Tomato is one of the most studied fruits, and both the plant and its fruit have been the subject of numerous studies. Functional compound extraction, mainly through environmentally friendly methods, remains an attractive research field for the utilisation of native tomato fruit, enhancing its limited production and use or harnessing the large amount of industrial waste from commercial tomato processing. There are few reports where clean extraction methods are combined; even rarer are those where green solvents are also used. In this work, the combination of different clean extraction technologies improved the extraction of carotenoids and tocopherols and allowed to establish a more efficient process. We believe that these results will stimulate the use of clean technologies and make the native tomato more attractive to harvest for industrial processing or to extract carotenoids and tocopherols for supplements.

Hydroethanolic Extract of Grape Peel from Vitis labrusca Winemaking Waste: Antinociceptive and Anti-Inflammatory Activities

Research background. Extracts from grape pomace, including the wine, showed many biological effects such as antioxidant and anti-inflammatory activities. Unfortunately winemakers discard the bagasse and the waste is less useful, however it contains bioactive compounds which result in antioxidant and anti-inflammatory properties. The work aimed to analyze the hydroethanolic extract of peels from agro-industrial waste from Vitis labrusca and to evaluate its antinociceptive and anti-inflammatory assays. This study is relevant for reusing a residue and adding value to the grape economic chain. Experimental approach. A representative sample of pomace was obtained and the peels were applied to produce the extract. The phenolic compounds were determined by multiple reaction monitoring mode of mass spectrometry methods and Folin-Ciocalteu, using gallic acid as standard. The biological analyzes were carried out using mice orally treated with crude extract at doses (30, 100, and 300 mg/kg). We evaluated mechanical hyperalgesia by the von Frey method, thermal heat hyperalgesia using a hot plate at 55 °C, paw edema using a pachymeter, and neutrophil recruitment by measurement of myeloperoxidase enzyme activity. The nephrotoxicity and hepatotoxicity were evaluated by biochemical analyses using blood samples that were collected after the Vitis labrusca administration. Results and conclusions. The peels correspond to 75 % of all wet winemaking residue and 59 % on a dry basis. We identified nine anthocyanins (3-O-glucoside: peonidin, delphinidin, petunidin, and malvidin; 3-p-coumaroyl-glucoside: cyanidin, peonidin, petunidin, and malvidin, and malvidin-3,5-diglucoside), five flavonoids (apigenin-7-glucoside, luteolin-7-glucoside, quercetin-3-galactoside, isorhamnetin-3-glucoside, and myricetin-3-rutinoside), and 26.62 mg GAE/g of phenolic compounds. In vivo assays, showed that Vitis labrusca extract at concentrations 100 and 300 mg/kg reduced carrageenan-induced mechanical and thermal hyperalgesia, 50 % of the paw edema, and neutrophil recruitment. In addition, there were no nephrotoxicity and hepatotoxicity. Our extract obtained from winemaking residue has analgesic and anti-inflammatory action, related at least in part to the presence of phenolic compounds, and it has no toxicity to renal and hepatic tissues. Novelty and scientific contribution. We demonstrated that this waste can be used for the production of antioxidant and anti-inflammatory products (pharmaceutical and cosmetics) without toxicity, contributing to the environmental economy.

Immunomodulatory Effect and an Intervention of TNF Signalling Leading to Apoptotic and Cell Cycle Arrest on ORL-204 Oral Cancer Cells by Tiger Milk Mushroom, Lignosus rhinocerus

Research background. Tiger milk mushroom (Lignosus rhinocerus) is a medicinal mushroom that is geographically distributed in the region of South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. Consumption of its sclerotium has been reported to treat various ailments. However, its anticancer potential towards oral cancer cell lines is yet to be discovered considering its traditional method of consumption by biting/chewing of the sclerotium. Experimental approach. Mushroom sclerotial powder of cultivar TM02® was extracted and fractionated by a Sephadex G-50 chromatographic column prior to cytotoxicity testing against a panel of human oral cancer cell lines. The capability of the identified bioactive fraction in regulating several molecules associated with its TNF pathway was investigated. Results and conclusions. MTT proliferation assay indicated that ORL-48 (derived from gingiva), ORL-188 (derived from the tongue), and ORL-204 (derived from buccal mucosa) were inhibited by L. rhinocerus sclerotial cold water extract and its high-molecular-mass fraction (HMM) in varying degree with ORL-204 being most affected. Hence, HMM treatment on ORL-204 was further investigated. HMM induced apoptosis and G0/G1-phase cell cycle arrest through caspase-3/7 cleavage. Activities of MIP2 and COX-2 were downregulated by 0.2- and 4.6-fold respectively in the HMM-treated ORL-204 cells. Novelty and scientific contribution. Using ORL-204, it revealed that HMM may have intervened via the TNF pathway at various network sites in its manifestation as a potential dietary compound for cancer prevention and natural adjunct therapeutic to conventional cancer treatment.

Effect of Process Variables on Rice Flour Functional Properties, and Porous Structure Properties of Rice and Wheat-Based Leavened Food Products

Research background. Various processing techniques impart significant impacts on physicochemical and functional properties of rice flour and the quality of the final products. This study aims to modify rice flour from different treatment combinations and to select the best treatment combination in developing rice-wheat-based leavened food products. Experimental approach. Eight treatment combinations were applied on rice flour according to 23 factorial design considering three variables at two levels namely, pre-treatment for rice grain modification [heat-moisture treatment, dual modification treatment (soaking rice grains in a NaHCO3 solution followed by heat treatment)], grinding technique (dry grinding, wet grinding), and flour particle size (75-180 µm, <75 µm). Eight dough samples were prepared by mixing 50 g of rice flour from each treatment with 50 g wheat flour. Thereafter, the dough samples were subjected to fermentation and gelatinization under pressurized condition (externally applied 1.0 kg/cm2 initial air pressure condition) in a pressure adjustable chamber. Results and conclusions. Results rendered that rice flour sample that underwent heat-moisture treatment followed by wet grinding and particle size at 75-180 µm, impart to improve the leavened gas retention capacity and obtaining highly porous and better textured rice-wheat based leavened food products under 1.0 kg/cm2 externally applied initial air pressure condition. Novelty and scientific contribution. Rice flour can be modified according to the present method to improve functional flour properties and the textural and structural properties of rice-wheat based leavened food products. Also, conducting fermentation and gelatinization under pressurized condition is a novel food processing technique, which contributes in restricting the escape of leavened gas from rice-wheat composite dough mass.

From the Autochthonous Grape Varieties of the Kastav Region (Croatia) to the Belica Wine

Research background. Coastal region of Croatia is rich in autochthonous grape varieties. Many of them have been almost abandoned such as the autochthonous varieties of Kastav (Croatia), used for the production of the Kastavska Belica wine. Therefore, the rationale of the presented study was to characterize autochthonous varieties 'Verdić', 'Mejsko belo', 'Jarbola', 'Divjaka' and 'Brajkovac' grape varieties. In addition, we performed a molecular characterization of the corresponding Belica wines. Experimental approach. Firstly, for five autochthons grape varieties, their genetic origin and ampelographic and economic characteristics were determined. Standard physico-chemical profiles and phenolic components by use of liquid chromatography coupled to triple quadrupole mass spectrometer (LC-QQQ) were determined for 12 corresponding wines from different producers. Fourier-transform infrared spectroscopy (FTIR) was used for determination of standard physico-chemical parameters Results and conclusions. Ampelographic analysis, which includes the data on producing characteristics and cluster and berry composition of varieties, revealed significant differences between analysed grape varieties. Results of physico-chemical analysis of the Belica wine showed that all wines met the requirements needed for the production of quality and top quality wines labelled with protected designation of origin (PDO) in Croatian coastal region. The LC-QQQ analysis confirmed the presence of different phenolic components in the Belica wines, where the most prominent phenols were flavonoids from the flavan-3-ol group. Overall, these results showed that autochthonous grapes from the Kastav region can be used for production of wines with added market value due to a growing demand for autochthonous products on the global market. Novelty and scientific contribution. The presented results represent scientific insight and a basis for further determination of the optimal cultivation technology aimed to take advantage of the best characteristics of each variety for production of a wine with desirable features.

Iron Encapsulation by Deacetylated Glucomannan as an Excipient Using the Gelation Method: Characteristics and Controlled Release

Research background. Deacetylation and the use of CaCl2 as a gelation agent improve the performance of glucomannan as iron encapsulant using the gelation method. This study was conducted to investigate the effects of deacetylation using NaOH and pH gelation on the characteristics of encapsulated iron using the CaCl2 gelation method. Experimental approach. Glucomannan was deacetylated at various NaOH concentrations and was subsequently utilized as an iron excipient using the pipette-dropped gelation method in CaCl2 solution to directly investigate the gelation process of encapsulation. The pH of the gelation solution was also changed. The beads were subsequently vacuum-dried. Results and conclusions. Deacetylation led to lower endothermic peak temperature of the glucomannan than that of the native one. The deacetylation degree (DD) and gelation pH did not significantly affect the diameter of the beads but influenced their appearance and physical characteristics. The backbone of glucomannan was not changed by either the deacetylation degree or the pH of the gelation treatment. The highest encapsulation efficiency (73.27 %) was observed in the encapsulated iron using the glucomannan matrix of the highest deacetylation degree (82.56 %) and gelated in pH=10 solution. The highest deacetylation degree of glucomannan caused the beads to have the highest swelling, which led to the release of a higher amount of iron. Glucomannan deacetylation improved the pH sensitivity of iron encapsulation, in which more iron was released at a pH=6.8 than of pH=1.2. The Weibull model was the best-fitted model to represent the profile of iron release from the deacetylated glucomannan matrix using the gelation method (R2 > 0.93) at pH=6.8 and pH=1.2 solutions. Novelty and scientific contribution. This result supports the application of deacetylated glucomannan using NaOH as a pH-sensitive matrix on iron encapsulation using CaCl2 solution as gelation agent. A higher deacetylation degree leads to the release of a higher amount of iron from the matrix. The encapsulation is not only protecting the iron but also delivering it to the absorption site and controlling the iron release which are useful in supplement formulation. or food fortifications. The results show that the deacetylated glucomannan as the matrix holds more iron in encapsulation process.

Prospects of Maize (Corn) Wet Milling By-Products as a Source of Functional Food Ingredients and Nutraceuticals

Maize (corn) consists of distinct parts, germ, endosperm, and pericarp, with different chemical compositions. During the maize wet milling process, the maize is disintegrated into the main product starch and by-products, including corn germ, corn fiber and corn gluten (the technical term for corn endosperm specific proteins and not the same as wheat gluten). These by-products are used as low-value animal feed products. The corn germ contains high amounts of tocols and phospholipids, while the corn gluten is rich in carotenoids and the corn fiber fraction is rich in phytosterols and complex carbohydrates. Each by-product has the potential to serve as a precursor in the manufacture of functional food ingredients or nutraceuticals that have antioxidant, anti-inflammatory, hypocholesterolemic, hypolipidemic and hypoglycemic properties. These food ingredients/nutraceuticals can be obtained through physical, chemical or enzymatic processes. Some nutraceuticals and food ingredients with market potential include corn fiber gum, oil, arabinoxylans, and xylo-oligosaccharides from corn fiber; corn germ oil and phospholipid ester from corn germ; and carotenoids and oligopeptides from corn gluten. This review focuses on current and prospective research into the use of corn germ, corn fiber and corn gluten in the production of potentially high-quality food ingredients or nutraceuticals.