Oilseed Extracts from Local Markets as Promising Coagulant Agents for Milk from Various Mammalian Species.

The aim of this study was to identify novel milk coagulants to be used in cheesemaking. For this purpose, aqueous extracts from safflower (Carthamus tinctorius), sunflower (Helianthus annuus), flax (Linum usitatissimum) and sesame (Sesamum indicum) seeds were tested for their caseinolytic (CA) and milk coagulating properties (MCA) in skim milk at temperatures of 25, 37, 50, 65 and 80 °C. The seed oil samples with the highest temperature ranges in regard to coagulation efficiency were then tested in cow, buffalo, goat and sheep milks and the MCA and curd yield (CY) parameters were measured at different temperatures. Due to their high milk coagulation efficiency (CE) in all types of milk and at different temperatures, the sesame and sunflower seed extracts proved to be particularly interesting and their CY parameters were similar to those obtained with animal rennet. Moreover, our results confirm that oilseed coagulants are capable of coagulating milk and can also be considered as potential animal rennet substitutes. This study provides valuable insights into the development of potential vegetable coagulants that could be used for various production processes aimed at specific target consumers.

Catalytic properties of lipoxygenase extracted from different varieties of Pisum sativum and Lens culinaris.

Lipoxygenase (LOX, E.C. 1.13.11.12), among its various roles, catalyzes the degradation of polyunsaturated fatty acids and it is considered to be one of the main causes of undesirable off-flavor developments in legumes. The role of LOX in postharvest physiology is particularly significant in seeds with high values of lipoxygenase and linoleic acid levels. This research aimed to study the biochemical properties of the LOX extracted from green pea (Pisum sativum L. var. Léda, Zeusz, Zsuzsi), dry pea (Pisum sativum L. var. Hanka, Irina, Lutra), and lentil (Lens culinaris L., var. Pinklevi, Rézi, Castelluccio), using linoleic acid as a substrate. The raw extracts showed different catalytic properties, with dry pea (var. Irina) that expressed the highest LOX activity, while lentil (var. Pinklevi) expressed the lowest activity. To complete the biochemical characterization of the crude LOX extracts, their optimal pH and temperature were also examined. The highest value of lipoxygenase activity in the pH range 6-7 was measured in all legumes. The optimal temperature for all extracts fell within the range of 30-60°C given the nutritional importance of legumes. This study will serve as a basis for further detailed investigation of the legumes LOX activity and its roles in food products related to legumes. PRACTICAL APPLICATIONS: This study investigated the biochemical properties of lipoxygenase (LOX) extracted from different varieties of lentil and pea, the two important leguminous crops serving as the main protein source for the population of humans worldwide. The biochemical properties of LOX extracted from legumes showed large differences in terms of kinetic properties. The results of this study revealed that the use of lipoxygenase can be a suitable index for managing stabilization techniques of lentil and pea, in order to inhibit the lipid oxidation in grain legume without compromising its nutritional value.

Heat and light stability of natural yellow colourants in model beverage systems.

The performance of 10 natural yellow colourants at different concentrations (0.001-0.3% w/v) was investigated in a model beverage system at various pH levels (3.5, 5.5 and 7.5) containing 10% w/v of sucrose, with or without ethanol (15% v/v) to simulate both alcoholic and non-alcoholic lemon-like beverages. Thus, the incidence of pH, ethanol and colourant concentration on spectral properties as well as turbidity were evaluated. The λmax was found to be independent from concentration in the tested range (0.001- 0.3% w/v), whereas the specific absorbance at λmax increased linearly. Moreover, the absorption spectrum and λmax of yellow pigments were found to be independent of pH and ethanol. Gardenia, safflower, and curcumin had the highest colour intensity and the lowest turbidity level. Heat and light stability were studied for these as well. Safflower was found to be the most stable colourant against heat (25, 40, 60, 80°C) and light (550 W/m2, 30°C).

An Evaluation of the Clotting Properties of Three Plant Rennets in the Milks of Different Animal Species.

Cynara cardunculus, Carica papaya and Ficus carica extracts are proposed as milk coagulants herein. Their coagulation efficiency was measured in bovine, buffalo, goat and sheep milk incubated at different temperatures. The milk-clotting and proteolytic activities as well as the lactodynamographic parameters were determined considering animal rennet as a reference coagulant. The vegetable coagulant, extracted from C. cardunculus pistils, proved to be the most suitable milk-clotting enzyme for cheesemaking, since it possesses similar milk clotting properties to conventional calf rennet. F. carica latex, but seemed to be a promising alternative coagulant at higher temperatures. The strong proteolytic activity of papain caused poor milk coagulation in all milk samples. To conclude, this result also supports the original hypothesis of this study that the excessive proteolytic nature of plant coagulants can negatively affect the cheesemaking process. The optimization of using a plant rennet in a dairy application can be done by selecting the appropriate plant rennet with a consistent clotting efficiency. These innovative manufacturing processes may also lead to the optimization and production of new cheese varieties.

Pre-fermentative cold maceration in presence of non-Saccharomyces strains: Evolution of chromatic characteristics of Sangiovese red wine elaborated by sequential inoculation.

Two different Metschnikowia strains (M. pulcherrima MP 346 or M. fructicola MF 98-3) were applied for the first time, during pre-fermentative cold maceration (PCM) in order to enhance the properties and stability of Sangiovese wine color. During the 2014 and 2015 vintages a total of eight wines were produced with 24 h of cold maceration (PCM 24 h) or 72 h (PCM 72 h), respectively. PCM was carried out in presence of MP 346 or MF 98-3 or pectic enzyme (Cuvée Rouge). The sequential inoculation of S. cerevisiae strain was carried out at the end of PCM. After 12 months in the bottle, the MP 346 and MF 98-3 wines contained much higher levels of total flavonoids than the Control sample for both vintages and regardless PCM duration. Moreover, in both vintages only MF 98-3 showed a higher color intensity than the Control sample after 12 months in the bottle. However, neither PCM duration nor the microbial/enzymatic treatment increased the level of anthocyanins at draining off. Both wines produced by the pre-fermentative inoculum with Metschnikowia strains (MP 346 and MF 98-3) retained their red hue, regardless the duration of pre-fermentative and fermentative macerations, while the Control wines were characterized by faster rates of color loss.

A preliminary study of continuous milk coagulation using Cynara cardunculus flower extract and calf rennet immobilized on magnetic particles.

The aim of this study was to develop a bioreactor design for continuous milk coagulation using a biocatalyst composed of immobilized animal and vegetable rennet on aminated magnetic particles, which has been proven to be an appropriate carrier for enzyme immobilization. Calf and vegetable (Cynara cardunculus) rennets were covalently immobilized on CLEA® magnetic supports and the immobilization procedure was optimized in batch mode, by evaluating protein loading, caseinolytic activity and the coagulation properties of skim milk powder and cow's milk. Subsequently the optimal temperature of immobilized coagulant was defined and a technically-friendly enzyme bioreactor was developed in order to carry out a continuous milk coagulation process with the aim of producing soft cheese.

Characterization of Active Dry Wine Yeast During Starter Culture (Pied de Cuve) Preparation for Sparkling Wine Production.

The preparation of yeast starter culture (Pied de Cuve) for producing sparkling wine with the traditional method is a key factor for manufacturing a good Prise de mousse. In this paper, the evolution of total yeast population, its viability during Pied de Cuve preparation, and the pressure profile during the 2nd fermentation in 2 different base wines made from Bombino bianco and Chardonnay grapes were investigated using 4 different commercial active dried yeasts. The study proves that despite the initial differences observed throughout the acclimatization phase, all the tested strains showed similar results on either the total population (from 8.2 × 10(7) cells/mL to 1.3 × 10(8) cells/mL) or cellular viability (from 70% to 84%). Independently from the base wine tested, the kinetic of sugar consumption was faster during the gradual acclimatization to the alcoholic medium (phase II) and slower during the preparation of starter culture in active growth phase (phase III). During both of these phases Saccharomyces cerevisiae bayanus Vitilevure DV10(®) (Station œnotechnique de Champagne) proved to have a higher sugar consumption rate than the other strains. During the Prise de mousse, S. cerevisiae bayanus Lalvin EC-1118(®) (Lallemand) reached the maximum pressure increase within time in both base wines.

Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation.

This research presents a feasibility study of using a continuous packed-bed reactor (PBR), containing immobilised native plant cysteine proteases, as a specific and mild alternative technique relative to the usual bentonite fining for white wine protein stabilisation. The operational parameters for a PBR containing immobilised bromelain (PBR-br) or immobilised papain (PBR-pa) were optimised using model wine fortified with synthetic substrate (Bz-Phe-Val-Arg-pNA). The effectiveness of PBR-br, both in terms of hazing potential and total protein decrease, was significantly higher than PBR-pa, in all the seven unfined, white wines used. Among the wines tested, Sauvignon Blanc, given its total protein content as well as its very high intrinsic instability, was selected as a control wine to evaluate the effect of the treatment on wine as to its soluble protein profile, phenolic composition, mineral component, and sensory properties. The treatment in a PBR containing immobilised bromelain appeared effective in decreasing both wine hazing potential and total protein amount, while it did not significantly affect the phenol compounds, the mineral component nor the sensory quality of wine. The enzymatic treatment in PBR was shown to be a specific and mild technique for use as an alternative to bentonite fining for white wine protein stabilisation.

Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study.

Lysozyme from hen egg white (HEWL) was covalently immobilized on spherical supports based on microbial chitosan in order to develop a system for the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. The objective is to limit the sulfur dioxide dosage required to control malolactic fermentation, via a cell concentration typical during this process. The immobilization procedure was optimized in batch mode, evaluating the enzyme loading, the specific activity, and the kinetic parameters in model wine. Subsequently, a bench-scale fluidized-bed reactor was developed, applying the optimized process conditions. HEWL appeared more effective in the immobilized form than in the free one, when the reactor was applied in real white and red wine. This preliminary study suggests that covalent immobilization renders the enzyme less sensitive to the inhibitory effect of wine flavans.

Assessing the Potential Content of Ethyl Carbamate in White, Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease.

The ethyl carbamate (EC) content of a wine after a given temperature-time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo-first-order kinetic rate constant for NH3 formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L(-1) . Such a sensitivity to TP was by far greater than that pertaining to a killed cell-based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors.

Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level.

Lysozyme immobilized on micro-sized magnetic particles: kinetic parameters at wine pH.

In order to use lysozyme as an anti-microbial agent during the winemaking process, hen egg-white lysozyme (LYZ) was covalently immobilized on two different micro-size magnetic particles (tosyl-activated and carboxylated, TSA and CA, respectively). A cell suspension of Oenococcus oeni, an oenological strain involved in the winemaking process, was utilized as LYZ substrate. Both a kinetic study and a study of the stability of free and immobilized LYZ were performed in McIlvane buffer at pH 3.2, that is the average minimum pH value in wine. The activity and kinetic parameters measured for the free LYZ at pH 3.2 are lower than those reported at the optimum pH (4.5); however the residual activity at pH 3.2 is sufficient to be of interest for further immobilization and applications in winemaking. All kinetic parameters of both biocatalysts (LYZ-CA and LYZ-TSA) are altered after immobilization, probably due to the structural modifications in the active site caused by covalent attachment to the supports. The half-life calculated at 25 °C was 39 h for free LYZ, while it increased to 280 and 134 h for LYZ-TSA and LYZ-CA, respectively. This result indicates that immobilization improves the enzyme stability and that LYZ can be utilized in wine applications in its immobilized forms. In addition, LYZ-TSA seems to be the best biocatalyst for further applications in winemaking.

Effect of wine inhibitors on free pineapple stem bromelain activity in a model wine system.

The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.