Tubular Cellulose Composite as a Vehicle for the Development of Meat Products with Low Nitrite Content.

Research background: Nitrite salts are among the most used preservatives in meat products as they ensure their safe consumption. Despite their positive effects on food safety and stability, many side effects on human health have been reported, leading to the need to reduce their use. Therefore, the aim of this study is to produce veal products with low nitrite content through low diffusion of potassium nitrite and to study their microbiological characteristics. Experimental approach: Edible tubular cellulose from leaf celery was produced and KNO2 was encapsulated in this material. This was achieved in two ways: by impregnation of tubular cellulose in a KNO2 solution under stirring and using starch gel as a stabilizer. Two samples of impregnated cellulose were applied on the surface of two veal samples of which one was stored at room temperature and the other at 3 °C. Similarly, two samples of cellulose with starch gel were applied on the surface of two veal samples of which one was stored at room temperature and the other at 3 °C. The KNO2 diffusion in different depths of the meat was measured and its effect on the microbiological characteristics of the meat was evaluated. Τhe experiment was carried out in duplicate. Results and conclusions: A satisfactory percentage of about 70 % of the initially encapsulated amount of KNO2 was diffused in the meat, while the rest remained in the pores of the delignified leaf celery. The migrating amount of KNO2 proved to be effective in preserving meat, as the microbiological load decreased significantly (especially within the first 12 h, from a decrease of 0.6 log CFU/g up to 2.4 log CFU/g). Novelty and scientific contribution: The demand for meat products with low nitrite content is constantly increasing and the results of the present study are promising for the development of this technology in scale-up systems and on an industrial scale. This innovative approach could lead to products with controlled diffusion of the preservatives.

Production and In Situ Modification of Bacterial Cellulose Gels in Raisin Side-Stream Extracts Using Nanostructures Carrying Thyme Oil: Their Physicochemical/Textural Characterization and Use as Antimicrobial Cheese Packaging.

We report the production of BC gels by Komagataeibacter sucrofermentans in synthetic (Hestrin and Schramm; HS) and natural media (raisin finishing side-stream extracts; RFSE), and their in situ modification by natural zeolite (Zt) and activated carbon (AC) nanostructures (NSs) carrying thyme oil (Th). The NS content for optimum BC yield was 0.64 g/L for both Zt-Th (2.56 and 1.47 g BC/L in HS and RFSE, respectively), and AC-Th (1.78 and 0.96 g BC/L in HS and RFSE, respectively). FTIR spectra confirmed the presence of NS and Th in the modified BCs, which, compared to the control, had reduced specific surface area (from 5.7 to 0.2-0.8 m2/g), average pore diameter (from 264 to 165-203 Å), cumulative pore volume (from 0.084 to 0.003-0.01 cm3/g), crystallinity index (CI) (from 72 to 60-70%), and crystallite size (from 78 to 72-76%). These values (except CI and CS), slightly increased after the use of the BC films as antimicrobial coatings on white cheese for 2 months at 4 °C. Tensile properties analysis showed that the addition of NSs resulted in a decrease of elasticity, tensile strength, and elongation at break values. The best results regarding an antimicrobial effect as cheese coating were obtained in the case of the RFSE/AC-Th BC.

Effect of cellulose crystallinity modification by starch gel treatment for improvement in ethanol fermentation rate by non-GM yeast cell factories.

This paper studies the reduction of crystallinity degree (CD) of cellulose treated with starch gel (SG), and the correlation of CD with the fermentation efficiency of cellulose to fuel-grade ethanol. Cellulose bioconversion from wood sawdust, consisting of three processes, was conducted in the same batch (one-step). The XRD and TEM analysis revealed 11% reduction in cellulose CD after its treatment with SG. One-step bioconversion process was performed employing two cell factories (CF) of non-engineered S. cerevisiae. CFs contained non- engineered S. cerevisiae cells covered with either SG entrapping Trichoderma reesei or cellulases prepared in the laboratory and immobilized in SG. The consolidated fermentation of treated cellulose resulted in an increase of bioethanol concentration (60-90%) in 2-day fermentation and the maximum ethanol concentration reached was approximately 5 mL/L (3.95 g/L). The fermentation efficiency for grade-fuel ethanol production was improved by cellulose pretreatment using SG to achieve reduced CD.

A critical review for advances on industrialization of immobilized cell Bioreactors: Economic evaluation on cellulose hydrolysis for PHB production.

Advances such as cell-on-cell immobilization, multi-stage fixed bed tower (MFBT) bioreactor, promotional effect on fermentation, extremely low temperature fermentation, freeze dried immobilized cells in two-layer fermentation, non-engineered cell factories, and those of recent papers are demonstrated. Studies for possible industrialization of ICB, considering production capacity, low temperatures fermentations, added value products and bulk chemical production are studied. Immobilized cell bioreactors (ICB) using cellulose nano-biotechnology and engineered cells are reported. The development of a novel ICB with recent advances on high added value products and conceptual research areas for industrialization of ICB is proposed. The isolation of engineered flocculant cells leads to a single tank ICB. The concept of cell factories without GMO is a new research area. The conceptual development of multi-stage fixed bed tower membrane (MFBTM) ICB is discussed. Finally, feasible process design and technoeconomic analysis of cellulose hydrolysis using ICB are studied for polyhydroxybutyrate (PHB) production.

Consolidated bioprocessing of lactose into lactic acid and ethanol using non-engineered cell factories.

The aim of this study is the consolidated bioprocessing of lactose into lactic acid and ethanol using non-engineered Cell Factories (CFs). Therefore, two different types of composite biocatalysts (CF1-CF2) based on Saccharomyces cerevisiae with immobilized microorganism or enzyme on starch gel (SG) were prepared for 5% w/v lactose fermentation. In CF1, S. cerevisiae was covered with SG containing Lactobacillus casei, Lactobacillus bulgaricus, Kluyveromyces marxianus CF1a-c. S. cerevisiae/SG-ß-galactosidase (CF1d) was also used for simultaneous saccharification and fermentation (SSF) of lactose. In CF2, S. cerevisiae immobilized on tubular cellulose (TC) was covered with SG containing the aforementioned microorganisms (CF2a-c). The wet CF1d resulted in 96% of the theoretical ethanol yield while the wet CF1b and freeze-dried CF2b resulted in 89% of the theoretical lactic acid yield. The repeated batches using the CF2a-c exhibited better results than using CF1a-c. Subsequently, the freeze-dried CF2 as preservative and more manageable were verified for future exploitation of whey.

Tubular Cellulose from Orange Juice By-Products as Carrier of Chemical Preservatives; Delivery Kinetics and Microbial Stability of Orange Juice.

The quality and safety of juices are assured mainly through heat treatments and chemical preservatives. However, there is a growing trend in the food industry for lowering energy and water demands, and the chemicals and additives that may have negative effects οn human health. Following that trend, in the present study, the reduced use of chemical preservatives in orange juice is proposed by using encapsulated sodium benzoate (SB) in tubular cellulose (TC), derived from orange pulp. The effects of SB concentration and contact time on SB encapsulation were evaluated. The use of the wet impregnation method, 12% w/v SB solution and 2 h of contact proved to be ideal for application in the juice industry. The use of starch gel resulted in a more stable composite (TC/SB-SG) with a slower SB delivery, showing its potential for future controlled delivery applications. Furthermore, similar delivery rates of SB in juice were noted at 25 and 2 °C. The TC/SB-SG proved capable of inhibiting the growth and reducing the numbers of spoilage microorganisms (yeasts and lactic acid bacteria). The results of the present study are promising for potential applications; however, more research is needed in order to evaluate the controlled delivery of SB in juice.

Cell factory models of non-engineered S. cerevisiae containing lactase in a second layer for lactose fermentation in one batch.

The objective of this project was to ferment lactose and whey to ethanol in one-step process. Models of cell factory of non-engineered S.cerevisiae have been proposed to ferment lactose. The cell factory of non-engineered S. cerevisiae/SG-lactase was prepared by the addition, of a starch gel solution containing lactase on non-engineered S. cerevisiae, and freeze drying of it. The 2-layer non engineered S.cerevisiae-TC/SG-lactase factory was prepared by immobilizing S. cerevisiae on the internal layer of tubular cellulose (TC), and the lactase enzyme was contained in the upper layer of starch gel (SG) covering cells of S. cerevisiae. Using such cell factory for the fermentation of lactose, alcohol yield of 23-32 mL/L at lactose conversion of 71-100%. The improvement in alcohol yield by cell factory versus co-immobilization of lactase enzyme and S. cerevisiae on alginates, was found in the range of 28-78%. Likewise, the cell factories are more effective than engineered S. cerevisiae. The fermentation of whey instead of lactose resulted in a significant reduction of the fermentation time. Freeze-dried cell factories led to improved results as compared with non-freeze dried. When lactase was substituted with L. casei, ethanol and lactic acid were produced simultaneously at high concentrations, but in a much longer fermentation time. The cell factories can be considered as models for white biotechnology using lactose containing raw materials. This suggested cell factory model can be applied for other bioconversions using the appropriate enzymes and cells, in the frame of White Biotechnology without genetic modification.

Entrapped Psychrotolerant Yeast Cells within Pine Sawdust for Low Temperature Wine Making: Impact on Wine Quality.

An alternative methodology is proposed for low temperature winemaking using freeze-dried raw materials. Pine sawdust was delignified and the received porous cellulosic material was applied as immobilization carrier of the psychrotolerant yeast strain Saccharomyces cerevisiae AXAZ-1. The immobilization of yeast cells was examined and verified by scanning electron microscopy (SEM). The immobilized biocatalyst and high-gravity grape must were separately freeze-dried without cryoprotectants and stored at room temperature (20-22 °C) for 3 months. The effect of storage on the fermentation efficiency of the immobilized biocatalyst at low temperatures (1-10 °C), as well as on the aromatic characteristics of the produced wines was evaluated. Storage time had no significant effect on the fermentation efficiency of the biocatalyst resulting in most cases in high ethanol production 13.8-14.8% v/v. The volatile fraction of the produced wines was examined using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography mass spectrometry (GC/MS). GC-MS/SPME analysis along with the organoleptic evaluation revealed in all produced wines a plethora of fresh and fruit aromatic notes. To conclude, fermentation kinetics and aromatic profile evaluation encourages the production of high-quality sweet wines at low temperatures using pine sawdust (Pinus halepensis) entrapped yeast cells as a promoter.

Freeze-dried immobilized kefir culture in cider-making.

BACKGROUND: The aim of the present study was to evaluate the fermentation efficiency of freeze-dried immobilized kefir culture on natural supports (apple pieces, delignified cellulosic material) in cider making at various temperatures (5-45 °C) in comparison with freeze-dried free cells. Freeze-dried cells were initially tested in apple juice fermentations at 30 °C, and then the freeze-dried cultures produced with no cryoprotectants were assessed in repeated batch fermentations. RESULTS: Repeated batch fermentations lasted for longer than 5 months. High malic acid conversion rates (up to 78.5%) and ethanol productivity values (up to 37.9 g L-1 day-1 ) were recorded for freeze-dried immobilized cells. Polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that freeze-drying had no effect on the microbial diversity of kefir culture. Higher alcohols were significantly reduced at low fermentation temperatures. Application of principal component analysis (PCA) revealed that both the fermentation temperature and the nature of the freeze-dried kefir culture affected significantly the minor volatiles determined by gas chromatography/mass spectrometry (GC/MS). Notably, all ciders produced were of high quality and were accepted by the tasting panel. CONCLUSIONS: Freeze-dried immobilized kefir culture on natural supports with no cryoprotectants was found to be suitable for simultaneous alcoholic and malolactic cider fermentation at various temperatures (5-45 °C). The high operational stability of the systems was confirmed and the results obtained are of great interest for the industrial sector as they could be exploited for cider, low-alcohol cider, or 'soft' cider production. © 2020 Society of Chemical Industry.

Enhanced Aromatic Profile and Functionality of Cheese Whey Beverages by Incorporation of Probiotic Cells Immobilized on Pistacia terebinthus Resin.

: In the present study, cheese whey was utilized for the development of a novel functional beverage, using Lactobacillus casei ATCC 393 probiotic cells immobilized on Pistacia terebinthus resin (pissa Paphos). Evaluation of shelf life of the produced beverages showed that spoilage microorganisms were not observed in beverages containing P. terebinthus resin. Terpenes' rich content might have contributed to the antimicrobial activity of the produced beverages; however, no significant effect on the viability of the immobilized probiotic cells was obtained. Whey beverages containing the immobilized biocatalyst retained a high viability (>1 × 106 CFU/g) of probiotic cells during a storage period of 30 days at 4 °C. The superiority of whey beverages containing the immobilized biocatalyst was also highlighted by GC-MS analysis, while the enhanced aromatic profile, which was mostly attributed to the higher concentration of terpenes, was also detected during the sensory evaluation performed. Conclusively, this study indicated the high commercialization potential of these novel functional whey beverages, within the frame of a sustainable dairy waste valorization approach. To the best of our knowledge, this is the first food-oriented approach within the guidelines of the circular economy reported in the literature, using the autochthonous Pistacia terebinthus resin for the production of functional whey beverages.

Effect of Sugar Content on Quality Characteristics and Shelf-Life of Probiotic Dry-Fermented Sausages Produced by Free or Immobilized Lactobacillus casei ATCC 393.

The aim of the present study was to investigate the effect of sugar content (0, 0.30, and 0.60%) on quality attributes and shelf-life of dry-fermented sausages stored for 66 days containing free or immobilized Lactobacillus casei ATCC 393 on wheat. For comparison, dry-fermented sausages with no starter culture were also produced. Physicochemical characteristics ranged within the levels usually observed in fermented sausages, while a drastic decrease was recorded in numbers of enterobacteria, staphylococci, and pseudomonads during ripening in all cases. Noticeably, sugar addition and the probiotic culture resulted in significant increase of shelf-life, whereas levels of L. casei ATCC 393 after 66 days of ripening persisted above 6 log cfu/g. Sugar addition had a positive effect on sensory attributes; although all products were of high quality, the immobilized cells provided a distinctive characteristic aroma and a fine taste.

Pistacia terebinthus Resin as Yeast Immobilization Support for Alcoholic Fermentation.

A natural resin retrieved from Pistacia terebinthus tree was evaluated as an immobilization carrier of Saccharomyces cerevisiae AXAZ-1 cells targeting successive fermentation batches of sugar synthetic mediums. Fermentation times below 54 h were recorded at temperatures 28-14 °C. In total, 147 compounds were detected using gas chromatography-mass spectrometry (GC-MS) analysis, including alcohols, esters, ketones, aldehydes, acids, and terpenes. Principal component analysis indicated that the state of cells (free/immobilized) and the fermentation temperature primarily affected terpenes' composition. Importantly, no spoilage of the fermented beverages was noted during 90 days of storage at room temperature, most likely due to the high content of extracted terpenoids and phenols (up to 579.01 mg L-1 and 171.8 mg gallic acid equivalent L-1, respectively). Likewise, the developed novel biocatalyst (yeast cells immobilized within Pistacia terebinthus resin) was suitable for the production of low alcohol beverages with an enhanced aromatic profile. The obtained results revealed that the proposed bioprocess shows great commercialization potential in the new fast-growing low-alcohol beverages sector.

Wine production using free and immobilized kefir culture on natural supports.

The aim of the present study was to perform simultaneous alcoholic and malolactic wine fermentations using free or immobilized kefir culture at a wide temperature range (5-45 °C). Repeated batch fermentations were carried out for a period up to 29 months, suggesting a high operational stability of the systems, while malic acid conversion and ethanol productivity up to 70.9% and 36.9 g/(Ld) were noted. Volatile acidity was at levels typically found in wines (<1.0 g acetic acid/L) in most cases, although increased values were recorded in wines produced at 5 °C, but no vinegar taint was detected. Fusel alcohols were at levels usually found in wines, but were reduced at 5 °C. Application of PCA to minor volatiles showed that the fermentation temperature rather than the nature of kefir culture had a significant effect. Noticeably, all products were accepted by the panel during the preliminary sensory evaluation.

Employment of L. paracasei K5 as a Novel Potentially Probiotic Freeze-Dried Starter for Feta-Type Cheese Production.

In the present study, a novel potentially probiotic Lactobacillus paracasei strain, previously isolated from dairy products, was evaluated as a starter culture of Feta-type cheese production. Targeting industrial applications, the starter culture was applied as a ready-to-use freeze-dried culture that was either free or immobilized. The immobilized biocatalyst composed of Lactobacillus paracasei K5 cells absorbed within delignified wheat bran prebiotic carrier. All produced cheeses were compared with cheese manufactured by renin enzyme. Several parameters that affect acceptability, quality and shelf-life of Feta-type cheese were investigated, including microbial populations, physicochemical characteristics and cheese volatiles through 90 days of ripening and storage. Survival of L. paracasei K5 remained in high levels (≥6.0 log cfu/g) after the 90th day of cheese production, as recorded by combining microbiological enumeration and strain-specific multiplex PCR analysis. The use of the freeze-dried novel starter culture (free or immobilized) enhanced the aromatic profile of Feta-type cheeses. Finally, the use of the potentially synbiotic immobilized biocatalyst further improved aromatic characteristics of produced cheese and decrease of possible spoilage or pathogenic microorganisms. These findings indicate the potential industrial use of freeze-dried L. paracasei K5 as starter culture for the production of good-quality functional Feta-type cheese.

Growth Capacity of a Novel Potential Probiotic Lactobacillus paracasei K5 Strain Incorporated in Industrial White Brined Cheese as an Adjunct Culture.

In this study, a novel potential probiotic strain Lactobacillus paracasei K5, previously isolated from traditional Greek Feta cheese and kefir grains, was evaluated as an adjunct culture for industrial white brined cheese production. Targeting industrial applications, apart from free cell cultures, a novel ready-to-use freeze-dried immobilized biocatalyst was prepared. The biocatalyst composed of L. paracasei K5 cells immobilized on delignified wheat bran prebiotic carrier and was freeze-dried without cryoprotectants. The adjunct free or immobilized culture was added separately without prior adaptation during white brined cheese manufacture and the produced cheeses were compared with commercial white brined cheeses. Several parameters that affect the acceptability and quality of the cheeses, including microbial populations, physicochemical parameters, volatile by-products and organoleptic characteristics, were analyzed through 70 days of storage. Results showed that the viability of the adjunct culture added either free or immobilized remained in high levels (7 to 8 log cfu/g) during maturation and storage. In addition, all white brined cheeses with the adjunct probiotic culture showed a sharp decrease in spoilage and pathogenic microorganisms such as enterobacteria, salmonella, staphylococci and coliforms during cheese maturation, especially when compared with the commercial white brined cheeses. Finally, after maturation time exceeded, all cheeses were characterized as safe for human consumption. Cheeses volatile compounds were significantly enhanced by the incorporated immobilized biocatalysts. These findings indicate the potential industrial use of freeze-dried ready-to-use immobilized lactobacilli as reinforcement adjunct cultures for the production of good-quality functional cheese products. PRACTICAL APPLICATION: The launch on market of novel foods developed by the incorporation of functional ingredients provides potential benefits to consumers' diet and new business opportunities for producers. Probiotic food products are one significant category of functional foods. Thus, this study focused on the development of a novel ready-to-use freeze-dried potential probiotic biocatalyst for functional white brined cheese production. The potential industrial application of such biocatalysts is highlighted by their incorporation as adjunct cultures that resulted in good-quality functional cheeses.

Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Technological Development of Brewing in Domestic Refrigerator Using Freeze-Dried Raw Materials.

Development of a novel directly marketable beer brewed at low temperature in a domestic refrigerator combined with yeast immobilization technology is presented in this study. Separately, freeze-dried wort and immobilized cells of the cryotolerant yeast strain Saccharomyces cerevisiae AXAZ-1 on tubular cellulose were used in low-temperature fermentation (2, 5 and 7 °C). The positive effect of tubular cellulose during low-temperature brewing was examined, revealing that freeze-dried immobilized yeast cells on tubular cellulose significantly reduced the fermentation rates in contrast to freeze-dried free cells, although they are recommended for home-made beer production. Immobilization also enhanced the yeast resistance at low-temperature fermentation, reducing the minimum brewing temperature value from 5 to 2 °C. In the case of high-quality beer production, the effect of temperature and initial sugar concentration on the fermentation kinetics were assessed. Sensory enrichment of the produced beer was confirmed by the analysis of the final products, revealing a low diacetyl concentration, together with improved polyphenol content, aroma profile and clarity. The proposed process for beer production in a domestic refrigerator can easily be commercialized and applied by dissolving the content of two separate packages in tap water; one package containing dried wort and the other dried immobilized cells on tubular cellulose suspended in tap water.

Scale-up for esters production from straw whiskers for biofuel applications.

Delignified wheat straw was fermented by a mixed bacterial anaerobic culture obtained from a UASB reactor to produce organic acids (OAs). Kissiris was used as immobilization carrier in a 2-compartment 82L bioreactor filled with 17L of fermentation broth for the first 7 fermentation batches and up to 40L for the subsequent batches. The amount of straw used was 30g/L and the temperature was set at 37°C for all experiments. The total OAs reached concentrations up to 17.53g/L and the produced ethanol ranged from 0.3 to 1mL/L. The main OAs produced was acetic acid (6-8g/L) and butyric acid (3-8g/L). The OAs were recovered from the fermentation broth by a downstream process using 1-butanol, which was the solvent with the best recovery yields and also served as the esterification alcohol. The enzymatic esterification of OAs resulted to 90% yield.

Downstream extraction process development for recovery of organic acids from a fermentation broth.

The present study focused on organic acids (OAs) recovery from an acidogenic fermentation broth, which is the main problem regarding the use of OAs for production of ester-based new generation biofuels or other applications. Specifically, 10 solvents were evaluated for OAs recovery from aqueous media and fermentation broths. The effects of pH, solvent/OAs solution ratios and application of successive extractions were studied. The 1:1 solvent/OAs ratio showed the best recovery rates in most cases. Butyric and isobutyric acids showed the highest recovery rates (80-90%), while lactic, succinic, and acetic acids were poorly recovered (up to 45%). The OAs recovery was significantly improved by successive 10-min extractions. Alcohols presented the best extraction performance. The process using repeated extractions with 3-methyl-1-butanol led to the highest OAs recovery. However, 1-butanol can be considered as the most cost-effective option taking into account its price and availability.

Production of nanotubes in delignified porous cellulosic materials after hydrolysis with cellulase.

In this study, tubular cellulose (TC), a porous cellulosic material produced by delignification of sawdust, was treated with a Trichoderma reesei cellulase in order to increase the proportion of nano-tubes. The effect of enzyme concentration and treatment duration on surface characteristics was studied and the samples were analyzed with BET, SEM and XRD. Also, a composite material of gelatinized starch and TC underwent enzymatic treatment in combination with amylase (320U) and cellulase (320U) enzymes. For TC, the optimum enzyme concentration (640U) led to significant increase of TC specific surface area and pore volume along with the reduction of pore diameter. It was also shown that the enzymatic treatment did not result to a significant change of cellulose crystallinity index. The produced nano-tubular cellulose shows potential for application to drug and chemical preservative delivery systems.