Transformation of agricultural wastes into functional oligosaccharides using enzymes and emerging technologies.

INTRODUCTION: Pectin-oligosaccharides (POS) serve diverse purposes as a food ingredient, antimicrobial and biostimulant in plants, and their functionality is linked to the degree of esterification. Grape and broccoli wastes emerge as environmentally friendly alternatives to obtaining pectin, serving as a sustainable source to producing POS. For example, microwaves have proven to be an effective and sustainable method to extract polysaccharides from plant matrices. OBJECTIVE: This work aims to use grape and broccoli wastes as alternative sources for obtaining pectin by microwave-assisted extraction and biotransformation into POS, which possess biological properties. MATERIAL AND METHODS: The extraction conditions were identified at a power of 400 W, 300 s for the extraction of pectin from grape pomace and broccoli waste. Biotransformation of pectins into POS, using commercial enzyme preparations (Viscozyme L and Pectinase). Characterisation was carried out by Fourier-transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. RESULTS: Physicochemical analysis indicated grape pomace and broccoli waste pectins had galacturonic acid content of 63.81 ± 1.67 and 40.83 ± 2.85 mg 100 mg-1, low degree of esterification of 34.89% and 16.22%, respectively. Biotransformation of pectins into POS resulted in a 20% hydrolysis rate. The main enzymatic activity was polygalacturonase for the degradation of the main structure of the pectin. CONCLUSION: Production of POS from agro-industrial wastes by emerging technologies, such as the combined use of microwave-assisted extraction and enzymatic processes, represents an alternative method for the generation of bioactive compounds with distinctive properties suitable for different applications of interest.

Scale-up and fed-batch cultivation strategy for the enhanced co-production of microbial lipids and carotenoids using renewable waste feedstock.

Agro-industrial by-product valorization as a feedstock for the bioproduction of high-value products has demonstrated a feasible alternative to handle the environmental impact of waste. Oleaginous yeasts are promising cell factories for the industrial production of lipids and carotenoids. Since oleaginous yeasts are aerobic microorganisms, studying the volumetric mass transfer (kLa) could facilitate the scale-up and operation of bioreactors to grant the industrial availability of biocompounds. Scale-up experiments were performed to assess the simultaneous production of lipids and carotenoids using the yeast Sporobolomyces roseus CFGU-S005 and comparing the yields in batch and fed-batch mode cultivation using agro-waste hydrolysate in a 7 L bench-top bioreactor. The results indicate that oxygen availability in the fermentation affected the simultaneous production of metabolites. The highest production of lipids (3.4 g/L) was attained using the kLa value of 22.44 h-1, while higher carotenoid accumulation of 2.58 mg/L resulted when agitation speed was increased to 350 rpm (kLa 32.16 h-1). The adapted fed-batch mode in the fermentation increased the production yields two times. The fatty acid profile was affected according to supplied aeration and after the fed-batch cultivation mode. This study showed the scale-up potential of the bioprocess using the strain S. roseus in the obtention of microbial oil and carotenoids by the valorization of agro-industrial byproducts as a carbon source.

Agroindustrial and food processing residues valorization for solid-state fermentation processes: A case for optimizing the co-production of hydrolytic enzymes.

In the pursuit of sustainability, managing agro-industrial and food processing residues (AFR) efficiently is crucial. This study proposes a systematic approach to convert AFR into valuable products via solid-state fermentation (SSF). Using fungal enzyme production as a case study, this adaptable methodology suits any SSF bioprocess. Initially, AFR's physicochemical properties were evaluated to assess their feasible use as carbon sources and solid matrices for SSF. Then, five strains were screened for their capability to produce enzymes (Xylanase, X; pectinase, P; cellulase, C). Apple pomace (AP) and brewery spent grain (BSG) with Aspergillus sp. (strain G5) were selected. Subsequent steps involved a two-phase statistical approach, identifying critical factors and optimizing them. Process conditions were screened using a Plackett-Burman design, narrowing critical variables to three (BSG/AP, pH, humidity). Response Surface Methodology (Central Composite Design) further optimized these factors for co-synthesis of X, P, and C. The humidity had the most significant effect on the three responses. The optimum conditions depended on each enzyme and were further validated to maximize either X, P or C. The obtained extracts were used for pectin extraction from orange peels. The extract containing primarily xylanase (X = 582.39, P = 22.86, C = 26.10 U mL-1) showed major pectin yield recovery (12.33 ± 0.53%) and it was obtained using the optimal settings of BSG/AP (81/19), humidity (50.40%), and pH (4.58). The findings will enable adjusting process conditions to obtain enzymatic cocktails with a tailored composition for specific applications.

Halophilic hydrolases as a new tool for the biotechnological industries.

Halophilic micro-organisms are able to survive in high salt concentrations because they have developed diverse biochemical, structural and physiological modifications, allowing the catalytic synthesis of proteins with interesting physicochemical and structural properties. The main characteristic of halophilic enzymes that allows them to be considered as a novel alternative for use in the biotechnological industries is their polyextremophilicity, i.e. they have the capacity to be thermostable, tolerate a wide range of pH, withstand denaturation and tolerate high salt concentrations. However, there have been relatively few studies on halophilic enzymes, with some being based on their isolation and others on their characterisation. These enzymes are scarcely researched because attention has been focused on other extremophile micro-organisms. Only a few industrial applications of halophilic enzymes, principally in the fermented food, textile, pharmaceutical and leather industries, have been reported. However, it is important to investigate applications of these enzymes in more biotechnological processes at both the chemical and the molecular level. This review discusses the modifications of these enzymes, their industrial applications and research perspectives in different biotechnological areas.

Co-microencapsulation: a promising multi-approach technique for enhancement of functional properties.

Co-microencapsulation is a growing technique in the food industry because it is a technique that, under the same fundamentals of microencapsulation, allows the generation of microcapsules with a longer shelf life, using a smaller number of encapsulating materials and a smaller amount of active compounds, while having a greater beneficial activity. This responds to consumer demand for higher quality foods that limit the use of ingredients with low nutritional content and provide beneficial health effects, such as probiotics, prebiotics, vitamins, fatty acids, and compounds with antioxidant activity. The combination of two or more active compounds that achieve a synergy between them and between the encapsulating materials offers an advantage over the well-known microencapsulation. Among the main active compounds used in this process are probiotics, prebiotics, fatty acids, and polyphenols, the main combination being that of probiotics with one of the other active compounds that enhances their benefits. The present review discusses the advantages and disadvantages of the different encapsulating materials and techniques used to obtain co-microencapsulants, where the main result is a higher survival of probiotics, higher stability of the active compounds and a more controlled release, which can lead to the generation of new foods, food supplements, or therapeutic foods for the treatment of common ailments.

Combined Pulsed Electric Field and Microwave-Assisted Extraction as a Green Method for the Recovery of Antioxidant Compounds with Electroactive Potential from Coffee Agro-Waste.

Coffee agro-waste is a potential source of polyphenols with antioxidant activity and application in the food and cosmetic trades. The usage of these byproducts persists as a challenge in the industrial landscape due to their high content of purported toxic substances hindering management. This study presents a green extractive process using pulsed electric field (PEF) and microwave assisted extraction (MAE) to recover polyphenols from coffee parchment and two varieties of pulp, posing quick processing times and the use of water as the only solvent. The performance of this process with regard to the bioactivity was assessed through the Folin-Ciocalteu assay, total flavonoid content, DPPH, ABTS and FRAP antioxidant tests. The phenolic composition of the extracts was also determined through HPLC-MS and quantified through HPLC-DAD. When compared to treatment controls, PEF + MAE treated samples presented enhanced yields of total phenolic content and radical scavenging activity in all analyzed residues (Tukey test significance: 95%). The chromatographic studies reveal the presence of caffeic acid on the three analyzed by-products. The HPLC-DAD caffeic acid quantification validated that a combination of MAE + PEF treatment in yellow coffee pulp had the highest caffeic acid concentration of all studied extraction methods.

Procyanidins: From Agro-Industrial Waste to Food as Bioactive Molecules.

Procyanidins are an important group of bioactive molecules known for their benefits to human health. These compounds are promising in the treatment of chronic metabolic diseases such as cancer, diabetes, and cardiovascular disease, as they prevent cell damage related to oxidative stress. It is necessary to study effective extraction methods for the recovery of these components. In this review, advances in the recovery of procyanidins from agro-industrial wastes are presented, which are obtained through ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, pressurized fluid extraction and subcritical water extraction. Current trends focus on the extraction of procyanidins from seeds, peels, pomaces, leaves and bark in agro-industrial wastes, which are extracted by ultrasound. Some techniques have been coupled with environmentally friendly techniques. There are few studies focused on the extraction and evaluation of biological activities of procyanidins. The identification and quantification of these compounds are the result of the study of the polyphenolic profile of plant sources. Antioxidant, antibiotic, and anti-inflammatory activity are presented as the biological properties of greatest interest. Agro-industrial wastes can be an economical and easily accessible source for the extraction of procyanidins.

Application of thermosonication for Aloe vera (Aloe barbadensis Miller) juice processing: Impact on the functional properties and the main bioactive polysaccharides.

The impact of thermosonication on the functional properties and the main polysaccharides from Aloe vera was investigated. Thermal processing was used for comparison purposes. Acemannan was the predominant polysaccharide in Aloe vera juice followed by pectins. Interestingly, thermosonication promoted a minor degradation of the acetylated mannose from acemannan than thermal processing. On the other hand, the degree of methylesterification of pectins was slightly reduced as a consequence of thermosonication. Further, swelling and fat adsorption capacities were improved by thermosonication. Thus, the highest values for swelling (>150 mL/g AIR) and for fat adsorption capacity (∼120 g oil/g AIR) were observed when thermosonication was performed at 50 °C for 6 min. Moreover, high inactivation of L. plantarum (∼75%) was observed when thermosonication was carried out at 50 °C for 9 min. Interestingly, thermosonication promoted a similar color change (ΔE = 7.7) to the modification observed during pasteurization carried out at 75 °C for 15 min (ΔE = 8.2 ±â€¯0.9). Overall, these results suggested that thermosonication could be a good alternative to thermal procedures of Aloe vera juice, since not only caused minor degradation of bioactive polysaccharides but was also able to improve functional properties.

Enzyme-Catalyzed Production of Potato Galactan-Oligosaccharides and Its Optimization by Response Surface Methodology.

This work shows an optimized enzymatic hydrolysis of high molecular weight potato galactan yielding pectic galactan-oligosaccharides (PGOs), where endo-ß-1,4-galactanase (galactanase) from Cellvibrio japonicus and Clostridium thermocellum was used. For this, response surface methodology (RSM) by central composite design (CCD) was applied. The parameters varied were temperature (°C), pH, incubation time (min), and enzyme/substrate ratio (U/mg). The optimized conditions for the production of low degree of polymerization (DP) PGOs were obtained for each enzyme by spectrophotometric assay and confirmed by chromatography. The optimal conditions predicted for the use of C. japonicus galactanase to obtain PGOs of DP = 2 were T = 51.8 °C, pH 5, E/S = 0.508 U/mg, and t = 77.5 min. For DP = 3, they were T = 21 °C, pH 9, E/S = 0.484 U/mg, and t = 12.5 min; and for DP = 4, they were T = 21 °C, pH 5, E/S = 0.462 U/mg, and t = 12.5 min. The efficiency results were 51.3% for substrate hydrolysis. C. thermocellum galactanase had a lower yield (35.7%) and optimized conditions predicted for PGOs of DP = 2 were T = 60 °C, pH 5, E/S = 0.525 U/mg, and time = 148 min; DP = 3 were T = 59.7 °C, pH 5, E/S = 0.506 U/mg, and time = 12.5 min; and DP = 4, were T = 34.5 °C, pH 11, E/S = 0.525 U/mg, and time = 222.5 min. Fourier transformed infrared (FT-IR) and nuclear magnetic resonance (NMR) characterizations of PGOs are presented.

Rapid physicochemical characterization of innovative fucoidan/fructan powders by ATR-FTIR.

Functional food has been highly demanded lately because of its benefits in counteracting diseases. Fucoidan and agave fructan are ingredients that enhance the growth of beneficial bacteria in the gut (prebiotics). This mixture has great potential to develop innovative products but it has never been explored before. Because of fucoidan is more expensive than agave fructan, the innovative proposed mixture is vulnerable to adulteration. This research was aimed to assess the accuracy of Fourier transform infrared spectroscopy with attenuated total reflectance (ATR-FTIR) coupled with chemometrics to identify and predict concentration of both polysaccharides in powder mixtures (0-100%). Absorption bands at 1240-1255 and 836-840 cm-1 were attributed to fucoidan and a strong peak at ~ 936 cm-1 confirmed the fructan presence. Peak areas were best fitted into linear models ( R adj 2  ≥ 0.92, RMSE ≤ 3.54%). This achievement may be useful to certificate ingredients contained in fucoidan-fructan mixtures, preventing adulteration.

Husk Tomato (Physalis ixocarpa Brot.) Waste as a Promising Source of Pectin: Extraction and Physicochemical Characterization.

Husk tomato (Physalis ixocarpa Brot. var. Rendidora) waste was evaluated as a source of specialized pectin, and pectin extracted from this waste was characterized physicochemically. Fruit was blanched for 10 or 15 min and extracted in 0.1 N HCl for 15 to 25 min. Extracted pectin was subjected to physicochemical analysis. For all extraction conditions, the percentage of anhydrogalacturonic acid exceeded 60%, indicating that husk tomato was a good source of pectin. The degree of esterification of pectin molecules was 63% to 91%. The amount of extracted pectin decreased with increasing extraction time. The apparent viscosity of husk tomato pectin showed the characteristic behavior of pseudoplastic fluids. Neutral sugars were identified, and the amounts of 6 sugars (fucose, rhamnose, arabinose, galactose, glucose, and xylose) were quantified. Sugars identified in husk tomato pectin and present in the Rhamnogalacturonan I region, arabinose, galactose, and rhamnose suggest a highly branched structure, which will influence its future applications. Molecular weight values were 542 to 699 kDa, exceeding molecular weight values reported for commercial citrus pectins from 134 to 480 kDa. The extraction process significantly (P < 0.05) influenced the physicochemical properties of pectin. Up to 19.8% from the total amount of pectin in the husk tomato was extracted by 10 min of blanching and 20 min of a more heat treatment. Our findings indicate that husk tomato can be a good alternative source of pectin having highly distinctive physicochemical characteristics.

Strategies to enhance the production of photosynthetic pigments and lipids in chlorophycae species.

Microalgae are a major natural source for a vast array of valuable compounds as lipids, proteins, carbohydrates, pigments among others. Despite many applications, only a few species of microalgae are cultured commercially because of poorly developed of cultivation process. Nowadays some strategies of culture have been used for enhancing biomass and value compounds yield. The most strategies applied to microalgae are classified into two groups: nutrimental and physical. The nutrimental are considered as change in media composition as nitrogen and phosphorous limitation and changes in carbon source, while physical are described as manipulation in operational conditions and external factors such as application of high-light intensities, medium salinity and electromagnetic fields. The exposition to electromagnetic field is a promising technique that can improve the pigments and biomass yield in microalgae culture. Therefore, is important to describe the advantages and applications of the overall process. The aim of this review was to describe the main culture strategies used to improve the photosynthetic and lipids content in chlorophyceae species.

Isolation and evaluation of tannin-degrading fungal strains from the Mexican desert.

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source.

Temperature model for process impact non-uniformity in genipin recovery by high pressure processing.

A model for the process impact temperature non-uniformity during high pressure processing (HPP) of genipap fruit purees was found during genipin recovery. Purees were subjected to HPP (130-530 MPa) under quasi-isobaric non-isothermal conditions (15 min; 0, 4.6 and 9.3mg pectinases/g fruit). Genipin and protein concentration was determined, and pH was measured. Polygalacturonase activity was quantified indirectly by protein content (mg/g fruit). First order kinetics described temperature changes (0-4 min). Polygalacturonase was activated at 130 MPa, inactivated reversibly at 330 MPa and activated again at 530 MPa. Enzyme reaction rate constant (k) was placed in the 0-4 min model and temperature from 2 to 15 min was described. Protein content and pH characterization in terms of decimal reduction time improved highly the 2-15 min model. Since temperature changes were modeled, more insight of its behavior in an HPP reactor was obtained, avoiding uniformity assumptions, making easier the industrial scale HPP implementation.

Immobilization of pectinesterase in genipin-crosslinked chitosan membrane for low methoxyl pectin production.

In this study, an environmentally friendly method for preparation of chitosan crosslinked membranes with different genipin concentrations (0, 125, 250, and 500 mg/L) was developed. Genipin-crosslinked chitosan membranes were used for the immobilization of fungal pectinesterase (PE). PE was efficiently immobilized in chitosan membranes and used for modification of high-methoxylated pectins into low-methoxylated pectins. The charge density (ζ-potential), infrared spectroscopy (FTIR), and ion-exchange chromatography (IEC) revealed the modification of pectin for immobilized PE in genipin-crosslinked chitosan membrane. The ζ-potential results indicated a decrease of -5.2 mV in the pectin control solution, whereas using the PE immobilized in chitosan and genipin-chitosan membranes, the obtained values were -30.45 mV and -36.38 mV, respectively. Genipin-crosslinked chitosan membrane jointly with the immobilized PE represents an eco-friendly support to prepare tailor-made low-methoxylated lime pectin.

Environmental friendly cold-mechanical/sonic enzymatic assisted extraction of genipin from genipap (Genipa americana).

An efficient cold-mechanical/sonic-assisted extraction technique was developed for extraction of genipin from genipap (Genipa americana) peel. Ultrasound assisted extraction (285 W, 24 kHz) was performed at 5, 10 and 15 °C for 5, 10 and 15 min. After cold-extraction, genipin was separated from pectin and proteins by aid of fungal pectinesterase. The maximum yield of non-cross-linked genipin was 7.85±0.33 mg/g, at 10 °C for 15 min by means of ultrasound extraction. The protein amount in extracts decreased in all samples. If mechanical process is combined with ultrasound assisted extraction the yield is increased by 8 times after the pectinesterase-assisted polyelectrolyte complex formation between pectic polysaccharides and proteins, avoiding the typical cross-linking of genipin. This novel process is viable to obtain non-cross-linked genipin, to be used as a natural colorant and cross-linker in the food and biotechnological industries.

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Background: Inulinases have been extracted and characterized from inulin-storing tissues; however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.

Purification and some properties of pectinesterase from potato (Solanum tuberosum L.) alpha cultivar

Pectinesterase was extracted from potato alpha cultivar, purified and partially characterized The used protocol resulted in a 58.8-fold purification (51 850.2 units/mg protein) with 15.5 percent recovery of pectinesterase activity. The purified enzyme had a molecular weight of 27 kDa and its isoelectric point was around 4.5 with pH and temperature optima of 8.0 and 60°C, respectively. The purified enzyme had a single symmetric peak of specific activity after chromatographic steps. The homogeneity of the purified pectinesterase was confirmed by gel filtration and polyacrylamide electrophoresis gel.

A pectinesterase foi extraída da batata (cultivar do alfa), purificada e parcialmente caracterizada. O protocolo usado levou a uma proteína purificada 58,8 vezes (51 850,2 units/mg da proteína) com uma recuperação de 15,5 por cento da atividade da proteína. A enzima purificada apresentou um peso molecular de 27 kDa e seu ponto isoelétrico foi ao redor 4,5. A pectinesterase exibiu pH e temperatura ótimos de respectivamente 8,0 e 60°C. A enzima purificada apresentou um único pico simétrico de atividade específica após as etapas de cromatografia. A homogeneidade da pectinesterase purificada foi confirmada por filtração em gel e por eletroforese em gel de poliacrilamida.