Juliano De Dea Lindner

Development of an Innovative Nutraceutical Fermented Beverage from Herbal Mate (Ilex paraguariensis A.St.-Hil.) Extract

Herbal mate (Ilex paraguariensis A.St.-Hil.) leaves are traditionally used for their stimulant, antioxidant, antimicrobial, and diuretic activity, presenting as principal components polyphenolic compounds. The aim of this work was to develop an innovative, non-dairy, functional, probiotic, fermented beverage using herbal mate extract as a natural ingredient which would also be hypocholesterolemic and hepatoprotective. Among different strains used, Lactobacillus acidophilus was selected as the best for fermentation. The addition of honey positively affected the development of L. acidophilus and the formulated beverage maintained microbial stability during shelf life. Key ingredients in the extract included xanthines, polyphenols and other antioxidants with potential health benefits for the consumer. Caffeine levels and antioxidant activity were also studied. Acceptable levels of caffeine and large antioxidant capacity were observed for the formulation when compared to other antioxidant beverages. An advantage of this product is the compliance to organic claims, while providing caffeine, other phyto-stimulants and antioxidant compounds without the addition of synthetic components or preservatives in the formulation. Sensorial analysis demonstrated that the beverage had good consumer acceptance in comparison to two other similar commercial beverages. Therefore, this beverage could be used as a new, non-dairy vehicle for probiotic consumption, especially by vegetarians and lactose intolerant consumers. It is expected that such a product will have good market potential in an era of functional foods.

A simple and fast method for the inspection of preservatives in cheeses and cream by liquid chromatography- electrospray tandem mass spectrometry

In this work, a simplified extraction and short time of analysis method for the simultaneous determination of natamycin, nisin and sorbic acid in cheeses and cream by reverse phase liquid chromatography-electrospray-tandem mass spectrometry was developed. Full validation was performed according to the Commission Decision 2002/657/EC criteria and method applicability was checked on several samples, aiming to inspect their compliance with regulatory limits. The method was linear in the concentration ranges of 0-10mg kg(-1) (natamycin), 0-25mg kg(-1) (nisin) and 0 20mg kg(-1) (sorbic acid). Samples of the three most consumed types of cheese (fresh, pasta filata and ripened) in Brazil and cream (ultra high temperature and pasteurized, 20-30% fat content) were assessed. A surprising rate of non-compliance was observed, especially among ripened grated cheeses, since 80% of samples were above the maximum limit for sorbic acid with an average concentration of 2766.3±10.8mg kg(-1). Moreover, a major non-compliance for the cream samples was observed. The proposed method can be applied as an efficient tool for the inspection of preservatives in cheeses and cream.

Development of a LC-MS/MS method for the simultaneous determination of sorbic acid, natamycin and tylosin in Dulce de leche.

A simple extraction, rapid routine method for the simultaneous determination of sorbic acid, natamycin and tylosin in Dulce de leche, a traditional South American product, by liquid chromatography-tandem mass spectrometry has been developed and fully validated. The limits of detection were set to 24.41mgkg(-1) (sorbic acid), 0.10mgkg(-1) (natamycin) and 2μgkg(-1) (tylosin). Recoveries ranged from 95% to 110%. Proportionally, internal standardization was more efficient than external standard, resulting in a smaller measurement of uncertainty. In total, 35 commercial samples from Brazil, Argentina and Uruguay have been assessed. The proposed method was tested on other dairy desserts, demonstrating to be versatile. Although tylosin was not detected in any sample, a high rate of non-compliance was found, with 67.39% of samples above the maximum allowed for sorbic acid and a maximum concentration of 2105.36±178.60mgkg(-1). In two samples, natamycin was irregularly found.

Diversity of Lactic Acid Bacteria Isolated from Brazilian Water Buffalo Mozzarella Cheese

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO2 from glucose. The diversity of LAB was evaluated by RAPD-PCR (randomly amplified polymorphic DNA-polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate-positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO2 from glucose and were citrate-positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in mozzarella cheese.

Biohydrogenation of Linoleic Acid by Lactic Acid Bacteria for the Production of Functional Cultured Dairy Products: A Review

Conjugated linoleic acid (CLA) isomers have attracted significant attention due to their important physiological properties, which have been observed in humans. Many lactic acid bacteria (LAB) demonstrate the ability to produce CLA isomers (C18:2 cis-9, trans-11 and C18:2 trans-10, cis-12) from the linoleic acid (LA) present in milk or in synthetic media. CLA isomers can be synthesized in vitro by LAB using vegetable oils rich in LA. The aim of this review is to present an update on the studies that have been conducted on the production of CLA isomers from LA mainly by LAB and of the factors that influence this conversion (source and concentration of LA and fermentation conditions). In addition, this review presents the relationship between the consumption of CLA isomers and their health benefits in humans such as anti-atherosclerosis and anti-carcinogenic effects. There is considerable variation between the studies concerning the beneficial effects of CLA in animal models, which have not been reflected in human studies. This can be attributed to the differences in the doses of CLA isomers used and to the different sources of CLA. Furthermore, the regulatory and scientific information classifying the physiological properties of CLA, which serve as support for the claims of its potential as a functional ingredient, are presented. More research is needed to determine whether CLA production by LAB can be enhanced and to determine the optimal requirements for these microbial cultures. Furthermore, safety and efficacy of CLA consumption have to be investigated in the future.

A multi-purpose tool for food inspection: Simultaneous determination of various classes of preservatives and biogenic amines in meat and fish products by LC-MS

This paper describes an innovative fast and multipurpose method for the chemical inspection of meat and fish products by liquid chromatography-tandem mass spectrometry. Solid-liquid extraction and low temperature partitioning were applied to 17 analytes, which included large bacteriocins (3.5kDa) and small molecules (organic acids, heterocyclic compounds, polyene macrolides, alkyl esters of the p-hydroxybenzoic acid, aromatic, and aliphatic biogenic amines and polyamines). Chromatographic separation was achieved in 10min, using stationary phase of di-isopropyl-3-aminopropyl silane bound to hydroxylated silica. Method validation was in accordance to Commission Decision 657/2002/CE. Linear ranges were among 1.25-10.0mgkg-1 (natamycin and parabens), 2.50-10.0mgkg-1 (sorbate and nisin), 25.0-200mgkg-1 (biogenic amines, hexamethylenetetramine, benzoic and lactic acids), and 50.0-400mgkg-1 (citric acid). Expanded measurement uncertainty (U) was estimated by single laboratory validation combined to modeling in two calculation approaches: internal (U = 5%) and external standardization (U = 24%). Method applicability was checked on 89 real samples among raw, cooked, dry fermented and cured products, yielding acceptable recoveries. Many regulatory issues were revealed, corroborating the need for enhancement of the current analytical methods. This simple execution method dispenses the use of additional procedures of extraction and, therefore, reduces costs over time. It is suitable for routine analysis as a screening or confirmatory tool for both qualitative and quantitative results, replacing many time consuming analytical procedures.

Development and application of a real-time polymerase chain reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas)

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method.

Development of a new analytical tool for assessing the mutagen 2-methyl-1,4-dinitro-pyrrole in meat products by LC-ESI-MS/MS

The use of sorbate and nitrite in meat processing may lead to the formation of 2-methyl-1,4-dinitro-pyrrole (DNMP), a mutagenic compound. This work was aimed at developing and validating an analytical method for the quantitation of DNMP by liquid chromatography-tandem mass spectrometry. Full validation was performed in accordance to Commission Decision 2002/657/EC and method applicability was checked in several samples of meat products. A simple procedure, with low temperature partitioning solid-liquid extraction, was developed. The nitrosation during the extraction was monitored by the N-nitroso-DL-pipecolic acid content. Chromatographic separation was achieved in 8 min with di-isopropyl-3-aminopropyl silane bound to hydroxylated silica as stationary phase. Samples of bacon and cooked sausage yielded the highest concentrations of DNMP (68 ± 3 and 50 ± 3 μg kg-1, respectively). The developed method proved to be a reliable, selective, and sensitive tool for DNMP measurements in meat products.

Effect of high-pressure carbon dioxide processing on the inactivation of aerobic mesophilic bacteria and Escherichia coli in human milk

ABSTRACT The effect of high-pressure carbon dioxide processing on inactivation of aerobic mesophilic bacteria and Escherichia coli ATCC 25922 inoculated in human milk was investigated. The effect of the ratio between sample mass and CO2 (1:0.2; 1:0.6 and 1:1 m/m); depressurization rate (1, 5.5 and 10 MPa/min); and pressure cycling (1, 3 and 5) were the process variables studied. The best reductions in aerobic mesophilic bacteria as well as in E. coli (>6.0 and >5.0 log, respectively) were obtained with a ratio of 1:1, a depressurization rate of 10 MPa/min, and one cycle of pressurization/depressurization. The depressurization rate was found to be an important variable in the inactivation process. The results suggest that high-pressure carbon dioxide processing can be applied to human milk as a safe alternative to the pasteurization employed in human milk banks.