Enzyme-Assisted Extraction for the Recovery of Food-Grade Chlorophyll-Based Green Colorant.

The aim of the study was to develop a biotechnological approach for the green recovery of chlorophyll from spinach, to be used as a natural food colorant. The plant matrix was characterized in terms of cell wall polysaccharide composition, and a tailored enzymatic mix based on cellulase (40%) xylanase (41%) and polygalacturonase (19%) was formulated. The process variables (temperature (°C), time (h), enzyme mix dose (U/g), zinc concentration (ppm), and buffer/substrate ratio (B/S)) and their interactions were studied by response surface methodology. The overlay plot made it possible to identify the process conditions (T: 25 °C, Zn: 150 ppm e B/S: 17.5, t: <2 h and enzyme mix dose between 12 and 45 U/g) to maximize the amount of chlorophyll, and concurrently, the quality of the green color of the extract. Finally, the novel colorant was applied in the production of a real food.

Partitioning Recovery of Natural Pigments from Spirulina platensis.

In the last few decades, the recovery of high-added value compounds with high food potential using microalgae as feedstock has been one of the main challenges for both research and industry. This study provides a simple, affordable, and cost-saving approach for the selective recovery of C-phycocyanin (C-PC), chlorophyll a and carotenoids from Spirulina platensis biomass by using biocompatible and industrially approved solvents (such as CaCl2 and ethanol). The concentration and yield of each pigment in the liquid extract have been spectrophotometrically detected, whereas the decolorized protein-rich biomass has been analyzed by CIELab parameters. The most concentrated (566.4 µg/mL) and food-grade C-PC extract (purity index 0.7) was obtained by applying a biomass/solvent ratio (1:10) for the first round of extraction (20 min), followed by a second round at 1:5 using CaCl2 1.5% (w/v) aqueous solution. Additionally, the same trial enabled the production of the brightest decolorized protein-rich biomass (L* = 46.2), characterized by a yellow-orange tonality (h° values = 81.3°).

Inclusion of curcumin in b-cyclodextrin: a promising prospective as food ingredient.

In the present work, the inclusion complexes between curcumin (CC) and ß-cyclodextrin (ß-CD) were obtained through co-precipitation at different times of magnetic stirring and simple mixing. The stoichiometric ratio between CC and ß-CD was ascertained to be 1:2. The samples prepared by co-precipitation (24 h magnetic stirring and 24 h cold static precipitation) and by simple mixing, showed the best performance in terms of powder recovery (72 and 97%, respectively), encapsulation efficiency (72 and 95%, respectively) and tinting power, while no significant differences were revealed in terms of loading capacity (about 14%). Using either inclusion method, the complexation of CC into ß-CD make the colorant 20-fold more water soluble than pure CC. The pigment stability was also enhanced under different storage conditions (high temperature and light exposure), either when applied in liquid or gel state. However, after 30 days under natural light exposure, an appreciable colour change from yellow to orange was revealed to an extent probably perceptible by an inexperienced observer.

A Minimally Invasive Approach for Preventing White Wine Protein Haze by Early Enzymatic Treatment.

Protein stability in bottled white wine is an essential organoleptic property considered by consumers. In this paper, the effectiveness of an early enzymatic treatment was investigated by adding a food-grade microbial protease at two different stages of winemaking: (i) at cold settling, for a short-term and low temperature (10 °C) action prior to alcoholic fermentation (AF); (ii) at yeast inoculum, for a long-lasting and medium temperature (18 °C) action during AF. The results reveal that protease sufficiently preserved its catalytic activity at both operational conditions: 10 °C (during cold settling) and 18 °C (during AF). Furthermore, protease addition (dosage 50-150 µL/L) raised the alcoholic fermentation rate. The treatment at yeast inoculum (dosage 50 µL/L) had a remarkable effect in preventing haze formation, as revealed by its impact on protein instability and haze-active proteins. This minimally invasive, time and resource-saving enzymatic treatment, integrated into the winemaking process, could produce stable white wine without affecting color quality and phenol content.

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.

Natural colorants from vegetable food waste: Recovery, regulatory aspects, and stability-A review.

Worldwide, approaches inspired by the Circular Economy model have been increasing steadily, generating new business opportunities such as the recovery of high-added value molecules (e.g., pigments) from vegetable food waste that may be applied as food additives (e.g., colorants). Indeed, food waste is a global problem that does not seem to be decreasing, leading to economic, environmental, and social issues. Moreover, synthetic dyes have been associated with adverse effects on human health, encouraging research to explore much safer, natural, and eco-friendly pigments. This state-of-the-art review gives a brief overview of the regulatory aspects concerning food waste, Circular Economy, and natural versus synthetic colorants. We have critically reviewed the recent advances in pigment recovery from vegetable food waste bringing back the green/unconventional extraction methods. Among them, enzyme-assisted extraction as a depth feature technique is highlighted, given that it allows the recovery of pigments in a mild, selective, efficient, and sustainable way. Furthermore, the stability issue of the different natural colorants has been critically discussed in relation to the extraction and application conditions. Several and tailored stabilization methods have been described and reported for each pigment although additional research is necessary on their long-lasting stabilization and utilization in food matrices. PRACTICAL APPLICATION: This review focuses on the main types of natural pigments in vegetable food waste, their legislative framework, extraction technologies and strategies to improve the stability , as well as their possible applications.

Phenolic-Degrading Enzymes: Effect on Haze Active Phenols and Chill Haze in India Pale Ale Beer.

The development of green and sustainable biotechnological approaches for preventing chill haze formation is currently under investigation. In this preliminary study, laccase and tannase (pure or combined) were applied as phenolic-degrading enzymes during two crucial brewing steps (i. post-mashing and ii. before the yeast inoculum). In post-mashing and irrespective of the dosage applied (100 µL/L or 1 mL/L), tannase-based treatment ensured the complete removal of haze active (HA) phenols, which was proved by the full prevention of chill haze (about 1 EBC vs. 22 EBC in the control sample). Before yeast inoculum for the alcoholic fermentation, the removal of haze active phenols and the prevention of chill haze were both tannase-dosage-dependent (15 and 2 EBC for the lowest and the highest dosages, respectively) although they failed to completely break down the HA phenols. This biotechnological approach did not significantly affect the chromatic properties of treated beer.

Betalain Extracts from Beetroot as Food Colorants: Effect of Temperature and UV-Light on Storability.

Beetroot is a good source of natural food colorants given that it contains significant amount of betalain pigments. This study investigates the three-months storage stability of betalain extracts recovered from unsold beetroot by a specific enzymatic mix. The extract storability was evaluated by the kinetic degradation model of betacyanins and betaxanthins, as well as by pigment color attributes (CIELa*b* parameters) under dark and UV-light exposure at different temperatures (4, 25 and 40 °C). Considering all the tested storage conditions, betaxanthins appeared to be more stable than betacyanins in terms of degradation rate constant, half-life time and activation energy. The effect of UV-light was negligible if the pigment was not exposed to temperatures higher than the room one, since no relevant differences were found between the parameters studied at 4 and 25 °C. Taking into account the visual color, all the investigated color indexes (total color difference, chroma and hue) were mainly affected by temperature, even if mildly high (40 °C), and to a lesser extent by UV-light exposure.

A Novel Process for the Recovery of Betalains from Unsold Red Beets by Low-Temperature Enzyme-Assisted Extraction.

Food waste management plays a central role in the circular economy. To our knowledge, only a few studies have investigated the use of unsold fruit and vegetables from supermarkets as a substitute source for the extraction of natural colorants. Thus, the aim of this paper was to suggest a green, tailored protocol that avoids the use of organic solvents for the recovery of betalains from unsold red beets for use as a food colorant. The recovery of such pigments was carried out by a tailored enzymatic mix, blended considering the polysaccharide composition of the beetroot cell wall; thus, it consisted of: cellulase (37%), xylanase (35%), and pectinase (28%). The enzyme-assisted extraction protocol was optimized, and the most suitable conditions (in terms of pigment yield and color attributes) for the recovery of betalains from unsold beets appeared to be: 25 U/g total dose of enzymatic mix, temperature 25 °C, and processing time 240 min.

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.

Novel microencapsulated yeast for the primary fermentation of green beer: kinetic behavior, volatiles and sensory profile.

The development of innovative and more cost-effective approaches of making beer throughout continuous fermentation process remains a challenging problem, which is worthy of serious exploration. The current work focuses on the application of a commercial brewing yeast (S. cerevisiae Nottingham Ale), entrapped into chitosan-calcium alginate double layer microcapsules, for the production of a Pale Ale beer. During the primary alcoholic fermentation, the consumption rate of fermentable brewing sugars and dissolved O2, estimated by the Gompertz equation, was halved in the beer obtained by encapsulated yeast in comparison with the free cell. The physical-chemical parameters of beer (i.e. pH, alcohol content, color and bitterness) were not remarkably affected by the different yeast-inoculating form. However, the volatile profiles identified by means of HS-SPME-GC-MS analysis, significantly differed in terms of terpenes, esters and alcohols content, thus proving that the yeast-inoculating form may typify the odor and flavor descriptors of the green beer.

Papain Covalently Immobilized on Chitosan-Clay Nanocomposite Films: Application in Synthetic and Real White Wine.

Increasing attention has been recently paid to the development of nanocomposite materials for food application as new tool to enhance the mechanical and thermal properties of polymers. In this study, novel chitosan-clay nanocomposite films were produced as carriers for the covalent immobilization of papain, by using a fixed amount of chitosan (1% w/v) and a food-grade activated montmorillonite (Optigel, OPT) or a high-purity unmodified montmorillonite (SMP), in four different weight percentages with respect to chitosan (i.e., 20, 30, 50, 70% w/w). Both nanoclays (OPT and SMP) improved the mechanical properties of the obtained nanocomposites, and the OPT films showed the highest Young modulus and mechanical resistance (σmax). The nanocomposites were used as carriers for the covalent immobilization of papain, which was preliminarily characterized in model wine towards a synthetic substrate, showing the highest efficiency in the release of the reaction product when it was bound on OPT-30 and OPT-50 films. Finally, the latter biocatalyst (papain on OPT-50 film) was applied for the protein stabilization of two different unfined white wines, and it efficiently reduced both the haze potential and the protein content.

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).

Evolution of phenolic and volatile compounds during bottle storage of a white wine without added sulfite.

BACKGROUND: Bottle storage can affect color, aroma and phenolic composition of white wine. Very little information is reported about the bottle evolution during storage of white wines without added sulfites and/or using other antioxidants. This work is aimed at studying the evolution of the main enological parameters, phenolic and volatile profiles of a white wine without added sulfites, during 15 months of storage in bottle at different light and temperature conditions, compared with a control stored with sulfur dioxide. Dark storage at 12 °C (D12-S, D12-NS) and 30 °C (D30-S, D30-NS) were compared to investigate the temperature effect, meanwhile uncontrolled temperature and light condition (UTL-S, UTL-NS) simulated improper storage conditions. RESULTS: Volatile acidity, absorbance at 420 nm and total phenols were higher in UTL-NS and D30-NS wines. The trans forms of hydroxycinnamic esters decreased, whereas, the cis forms, as well as caffeic acid derivatives, significantly increased in samples without added sulfites. The storage without sulfites also accelerated the hydrolysis of acetate esters. However, it did not affect most of the ethyl esters whose content remained almost the same between sulfite added (S) and no sulfite added (NS) wines. CONCLUSION: The presence of sulfites in the bottle helped to preserve the volatile compounds of young wines; however, even more important seemed to be the optimal storage (dark and low temperature), as unsuitable conditions favored aroma degradation of bottled wine, regardless of sulfite protective action. © 2019 Society of Chemical Industry.

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.

Yeast cells in double layer calcium alginate-chitosan microcapsules for sparkling wine production.

This paper focuses on the use of a new type of yeast encapsulation procedure, applying the chitosan-calcium alginate double layer microcapsules, for the production of Riesling sparkling wine. Four different sparkling wines were produced by free or encapsulated yeasts. The four types of yeast used were adapted (Free EtOH-A, Encapsulated EtOH-A) and non-adapted to ethanol (Free, Encapsulated). The different yeast-inoculating formats had a significant impact on oxygen consumption and pressure increase rate in the bottle during the prise de mousse. Similarly to the free form, encapsulated yeast successfully completed the secondary fermentation. After an ageing period of 6 months, volatiles and sensory profiles of sparkling wines were compared. Although, some differences in volatile profiles were found among samples, sparkling wines produced by Encapsulated EtOH-A showed sensory properties, in terms of aroma, taste and body, similar to those produced by free yeast (both adapted and non-adapted to ethanol).

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.