Clotting and Proteolytic Activity of Freeze-Dried Crude Extracts Obtained from Wild Thistles Cynara humilis L. and Onopordum platylepis Murb.

The rising interest in finding alternatives to animal rennet in cheese production has led to studying the technological feasibility of using and exploiting new species of herbaceous plants. In this research work, and for the first time, freeze-dried extracts from Cynara humilis L. (CH) and Onopordum platylepis Murb. (OP) were studied for mineral and protein content, and their clotting and proteolytic activity were compared to those of Cynara cardunculus L. (CC). The effect of extract concentration (5-40 mg extract/mL), temperature (20-85 °C), pH (5-8), and CaCl2 concentration (5-70 mM) on the milk clotting activity (MCA) of CC, CH and OP extracts was evaluated. The MCA values were significantly higher in CC at the same extract concentration. The extract that showed the most significant increase in clotting activity due to increased temperature was OP, with maximum activity at 70 °C. The pH value for maximum milk clotting was 5.0 for both CC and CH, whereas, in the case of OP, the pH value was 5.5. CaCl2 enhanced the clotting capacity of the extracts, particularly for OP and CH. Furthermore, proteolytic activity (PA) and the hydrolysis rate increased with increasing time and enzyme concentration, with CC being the extract that achieved the highest caseinolytic activity.

Effects of time and temperature of storage on chemical and nutritional characteristics of raw milk for Provolone Valpadana PDO cheesemaking: a multivariate approach.

We evaluated the possibility of increasing the storage temperature of raw milk for Provolone Valpadana cheesemaking, to identify the most suitable conditions of time and temperature for a pre-maturation process. We used Principal Component Analysis (PCA) to analyze the overall effects of different storage conditions on chemical, nutritional and technological characteristics of the raw milk. Four different thermal storage cycles, two at fixed temperature/time (6 and 12°C for 60 h) and two with two-phase thermal cycle (10 and 12°C for 15 h, followed by refrigeration at 4°C for 45 h) were studied. Although a moderate heterogeneity among raw milks from the 11 producers of Provolone Valpadana cheese was observed, PCA revealed the critical aspects of the extreme storage conditions (60 h of refrigeration). Some samples resulted in anomalous behaviors, probably related to unexpected fermentation phenomena occurring with increasing storage temperature. The acidification and the increase in the contents of lactic acid, soluble calcium, and degree of retinol isomerization observed in the anomalous samples can compromise the technological functionality of milk. Conversely, the storage with a two-phase thermal cycle did not lead to variations in any measured characteristic, suggesting that mild refrigeration conditions (10 or 12°C for 15 h followed by 4°C for 45 h) could be a good compromise in favoring milk pre-maturation without altering its quality characteristics.

Potentialities of aqueous extract from cultivated Onopordum tauricum (Willd.) as milk clotting agent for cheesemaking.

In Western and Central Mediterranean countries proteases from wild herbaceous perennial plants commonly known as "thistles" have been used as milk coagulants in cheese-making for centuries. For the first time, the technological and biochemical traits of proteases from cultivated Onopordum tauricum Willd. (Taurian thistle, bull cottonthistle) were assessed. The optimal conditions for minimizing the clotting time and the non-specific proteolytic activity were estimated at the highest (T = 43-45 °C; [Ca2+] = 11-13 mM) and the lowest (T = 35-39 °C; [Ca2+] = 5 mM) temperature and calcium ion levels in the explored range respectively, thus highlighting the difficulty to set the best operative compromise in the first step of cheesemaking. In the conditions adopted in common cheesemaking practice (T = 37 °C; pH = 6.5) 1 mL of reconstituted extract from cultivated thistles coagulated 10 mL of ewe's and goat's milk in 114-146 and 129-167 s, respectively, and 1 mL of reconstituted extract from spontaneous thistles coagulated 10 mL of ewe's and goat's milk in 232-294 and 428-621 s, respectively, while no significant differences in the non-specific proteolytic activity between cultivated and spontaneous O. tauricum extracts were observed. The purified enzyme (tauricosin) was identified as an aspartic protease made up of two sub-units with molecular weights of 32 and 9.6 kDa, respectively. Experimental data encouraged the exploitation of O. tauricum as a new and sustainable non-food crop in marginal and rainfed lands of Mediterranean countries, thus reducing the potential biodiversity losses due to wild collection.

Cow and Ewe Cheeses Made with Saffron: Characterization of Bioactive Compounds and Their Antiproliferative Effect in Cervical Adenocarcinoma (HeLa) and Breast Cancer (MDA-MB-231) Cells.

Saffron is a widespread consumed spice containing many phytochemicals. It is often used in dairy technologies to enhance color and flavor of cheeses, but it is also known for its several therapeutic effects, as well as its antiproliferative and anticancer properties. In this study High Performance Liquid Chromatography was used to characterize saffron bioactive compounds in cow and ewe cheeses made with saffron, and the antiproliferative effect of the crocin-rich extracts from cheeses was investigated on different cellular lines (CaCo2, MDA-MB-231 and HeLa) by MTT assay. Crocins were observed in all cheese samples, with the total content ranging between 0.54 and 30.57 mg trans-4-GG/100 g cheese, according to the different cheese making process. Picrocrocin was detected in no cheese (probably due to its degradation during cheese making), while safranal was detected only in one ewe cheese (mainly due to its high volatility). HeLa and MDA-MB-231 cells were sensitive to treatment with crocin-rich extracts from cheeses, while no effect was observed on CaCo2 cells. The chemical environment of the food matrix seems to have a great influence on the crocin antiproliferative effect: the crocin-rich extracts from cheese with both high residual N/protein and fat contents showed increased antiproliferative effect compared to pure crocin (trans-4-GG), but cheeses from different milk species (type of fats and proteins) could also play an important role in modulating crocin's antiproliferative effects.

Content and Nutritional Evaluation of Zinc in PDO and Traditional Italian Cheeses.

Zinc is an essential mineral which plays a key role in several important biological processes in the human body. The determination of its level in food matrices can contribute to the food quality characterization and to the adequacy of the diet. Animal food products generally have a higher zinc content compared to vegetables. Among them, dairy products consumption can provide a great contribution to the zinc reference intakes. In this study, different Italian cheeses (38 Protected Denomination of Origin and 9 Traditional) were evaluated for their zinc content. Cow cheeses generally showed the highest zinc content (1.83-7.75 mg/100 g cheese), followed by sheep cheeses (1.34-3.69 mg/100 g), and cheeses from mixed milk (0.39-4.54 mg/100 g). The only cheese from buffalo milk (Mozzarella di Bufala Campana PDO) showed a zinc content of 2.14 mg/100 g. The great variability in the zinc content observed among the samples is the result of the influence of several factors, such as the feeding system, the species (cow, sheep, goat, and buffalo), and the cheese-making. Most of the samples resulted in a great contribution (>10%) to the zinc Daily Reference Intake set by EU (10 mg/day), with only two samples contributing to less than 4%.

Rapid Determination of Total Tryptophan in Yoghurt by Ultra High Performance Liquid Chromatography with Fluorescence Detection.

Tryptophan (TRP) is an essential amino acid which cannot be synthesized by humans and animals, but has to be supplied by exogenous sources, notably through the diet. The bulk of dietary TRP flows into the synthesis of body's proteins, but the TRP metabolism also involves several biochemical reactions (i.e., serotonin and kynurenine pathways). Defects in the TRP transport mechanism or catabolism are related to a large number of clinical abnormalities. Therefore, dietary TRP intake is necessary not only for the body's growth but also for most of the body's metabolic functions. Among protein-based foods, milk proteins provide a relatively high amount of TRP. In this paper, a rapid chromatographic method for TRP determination in yoghurt, by ultra high performance liquid chromatography on a reversed-phase column with fluorescence detection (280 nm Ex; 360 nm Em), is provided. A linear gradient elution of acetonitrile in water allowed TRP analysis in 8.0 min. The limit of detection and limit of quantification of the method were 0.011 ng/µL and 0.029 ng/µL, respectively, using 5-methyl-l-tryptophan as the internal standard. The analytical method was successfully applied to commercial yoghurts from different animal species, and the TRP values ranged between 35.19 and 121.97 mg/100 g (goat and cow Greek type yoghurt, respectively).

Natural Preservatives from Plant in Cheese Making.

Today, consumers are increasingly demanding safety alternatives concerning the use of synthetic additives in the food industry, as well as healthy food. As a result, a major number of plant-derived preservatives have been tested in the food industry. These natural ingredients have antioxidant properties and have shown to increase the bioactive molecules levels and the microbiological stability of the food items. The effect of the plant-based preservatives on the sensorial properties of the new products has also to be considered, because natural preservatives could result in sensorial characteristics that may not be accepted by the consumers. Cheese is a dairy product widely appreciated all over the world, but it is also susceptible to contamination by pathogenic and spoilage microorganisms; therefore, the use of preservatives in cheese making represents an important step. This review deals with one of the innovation in the cheese sector, which is the addition of natural preservatives. Several aspects are discussed, such as the effect of natural ingredients on the microbial stability of cheese, and their influence on the chemical, nutritional and sensorial characteristics of the cheeses. Although the promising results, further studies are needed to confirm the use of natural preservatives from plants in cheese making.

New Trends for the Evaluation of Heat Treatments of Milk.

Milk is generally very rich in nutrients and this may lead it to be an ideal growth environment for many microorganisms, including pathogens, so effective measurements aiming to ensure total microbiological safety of milk and minimize the risk to human health are needed. Milk heat treatments are the most common practices carried out to inhibit the microbial growth; therefore it is necessary to have analytical procedures that are more and more up-to-date and capable of detecting the effectiveness of the heat treatments. Most of the reference and official methods to assess heat treatment in milk are based on the evaluation of the modifications of some milk components following the thermal process, such as the determination of enzyme activities (alkaline phosphatase and lactoperoxidase), whey proteins, Maillard reaction compounds (generally furosine), and lactulose. Besides the most common techniques (liquid and gas chromatography, capillary electrophoresis, or spectroscopy) used for the detection of single thermal indicators, new approaches, such as chemometric studies or more recent techniques, including size-exclusion chromatography with online electrospray mass spectrometry or stable isotope ratio mass spectrometry, are discussed in this review in order to evaluate heat treatment in milk.

Qualitative Study of Functional Groups and Antioxidant Properties of Soy-Based Beverages Compared to Cow Milk.

Soy-based beverages are a source of high quality proteins and balanced nutrients; they thus represent an alternative to milk in case of allergy to cow milk proteins or intolerance to lactose. In this research, antioxidant properties of soy-based beverages and UHT cow milk were studied. In addition, color parameters, by a quick and non-destructive methodology, were studied in order to verify a possible correlation with antioxidant properties and a qualitative analysis of the major functional groups undertaken by Fourier Transformed Infrared Spectroscopy (FTIR) on Attenuated Total Reflectance (ATR) was carried out. Extractable and hydrolysable polyphenols were studied in soy-based beverages. However, only the extractable fraction was studied in UHT milk, which was characterized by a small amount of polyphenols. All color parameters showed highly significant differences among soy-based beverages and between soy-based beverages and cow milk. FTIR-ATR spectra of soy-based beverages and cow milk showed several differences in the various regions depending on both the specific contribution of molecular groups and different food items.

Updating Nutritional Data and Evaluation of Technological Parameters of Italian Milk.

Different technologically treated Italian milks (whole and semi-skimmed ultra-high temperature (UHT), pasteurized and microfiltered milk), collected from 2009 to 2012, were evaluated for nutritional and technological properties. No significant differences in calcium and sodium were detected (p > 0.05), while significant differences were observed concerning phosphorus content, between whole and semi-skimmed milk, and lactose content, between pasteurized and UHT milk (p < 0.05). In UHT milk, lactose isomerization occurred, and lactulose (from 8.6 to 104.0 mg/100 g) was detected. No significant differences (p > 0.05) were detected for choline, a functional molecule, between whole (11.3-14.6 mg/100 g) and semi-skimmed milk (11.1-14.7 mg/100 g), but there were significant differences (p < 0.05) in processing milk (UHT vs. pasteurized milk and UHT vs. microfiltered milk). Among the unsaponifiable compounds, only 13 cis retinol and trans retinol showed differences in technologically treated milk (pasteurized vs. UHT milk and microfiltered vs. UHT milk; p < 0.05). In this research, the greater was the "severity" of milk treatment, the higher was the percent ratio 13 cis/trans retinol (DRI, degree of retinol isomerization). The degree of antioxidant protection parameter (DAP), useful to estimate the potential oxidative stability of fat in foods, was significantly different between whole and semi-skimmed milk (p < 0.05). Finally, the evaluation of color measurement of whole milk showed a good correlation between beta carotene and b* (r = 0.854) and between lactulose and a* (r = 0.862).

Taurine in milk and yoghurt marketed in Italy.

Taurine, a free amino acid, was studied as natural compound of different typologies of milk: pasteurized, ultra-high temperature (UHT), microfiltered whole and semi-skimmed cow's milk; pasteurized and UHT goat's whole milk and raw buffalo's whole milk. Moreover, taurine contents in yoghurt from cow and goat's milk were evaluated. The data obtained in this research showed that no significant variations of taurine occurred in cow's milk subjected to different technological processes and between whole and semi-skimmed milk. The amount of taurine was less (p < 0.05) in cow's milk (0.60 mg/100 g) than in goat and buffalo's milk (6.55 and 7.32 mg/100 g, respectively). No significant differences in taurine occurred between goat and buffalo's samples. The amounts of taurine in yoghurt reflected, substantially, the content of this molecule in the milk of the relevant animal species. These results are noteworthy because data available in the literature on this molecule in commercial dairy products are old or few.

Kinetic study on unsaponifiable fraction changes and lactose hydrolysis during storage of Mozzarella di Bufala Campana PDO cheese.

Cholesterol, vitamin E and vitamin A isomers, components of the unsaponifiable fraction, and lactose contents in Mozzarella di Bufala Campana PDO cheese have been studied during storage in different conditions: at 6-8 degrees C, and at room temperature (traditional habits). During cold storage there were no significant differences in the unsaponifiable compounds, while during storage at room temperature an increase of the 13-cis isomer and a decrease of all-trans-retinol were observed. Trans/cis-retinol isomerization, expressed as the Degree of Retinol Isomerization (13-cis/all-trans%) showed an increase in all samples during storage at room temperature following a linear correlation. The decrease of lactose content during storage was studied by a first-order differential relaxation equation. Apart from the traditional habits, consumers should be aware that, during storage at room temperature, the most important changes in the considered nutrients occur. These changes can be negative (vitamin A reduction) but also positive (lactose reduction).