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.

Optimization of bacterial cellulose production by Komagataeibacter sucrofermentans in synthetic media and agrifood side streams supplemented with organic acids and vitamins.

The effect of thiamine (TA), ascorbic acid (AA), citric acid, and gallic acid (GA) on bacterial cellulose (BC) production by Komagataeibacter sucrofermentans, in synthetic (Hestrin and Schramm, HS) and natural substrates (industrial raisins finishing side stream extract, FSSE; orange juice, OJ; green tea extract, GTE), was investigated. The Response Surface Methodology was found reliable for BC yield prediction and optimization. Higher yields were achieved in the FSSE substrates, especially those supplemented with AA, TA, and GA (up to 19.4 g BC/L). The yield in the non-fortified substrates was 1.1-5.4 and 11.6-15.7 g/L, in HS and FSSE, respectively. The best yield in the natural non-fortified substrate FSSE-OJ-GTE (50-20-30 %), was 5.9 g/L. The porosity, crystallinity, and antioxidant properties of the produced BC films were affected by both the substrate and the drying method (freeze- or oven-drying). The natural substrates and the process wastewaters can be further exploited towards added value and sustainability. Take Home Message Sentence: Raisin and citrus side-streams can be efficiently combined for bacterial cellulose production, enhanced by other vitamin- and phenolic-rich substrates such as green tea.

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.

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.

Vinegar Production from Corinthian Currants Finishing Side-Stream: Development and Comparison of Methods Based on Immobilized Acetic Acid Bacteria.

Fruit wastes and side-streams can be used for vinegar production to create added value for the agri-food sector and enhance farmer incomes and local economies. In this study, methods for vinegar production by wild and selected acetic acid bacteria (the quick starter Acetobacter aceti and the acid-resistant Komagataeibacter europaeus), free (FC) and immobilized (IC) on a natural cellulosic carrier, are proposed using sweet wine made from the industrial finishing side-stream (FSS) of Corinthian currants as raw material. The results showed all cultures can produce vinegar with 46.65 ± 5.43 g/L acidity, from sweet FSS wine containing 5.08 ± 1.19% alcohol. The effect of immobilization was more obvious in the case of the selected culture, presenting better acetification efficiency, both fresh and after cold storage for 2 months. The vinegars had an antioxidant capacity of 263.5 ± 8.4 and 277.1 ± 6.7 mg/L (as ascorbic acid) and phenolic content 333.1 ± 12.0 and 222.2 ± 2.9 mg/L (as gallic acid) (for FC and IC, respectively). They also had a rich volatilome (140 compounds identified by SPME GC-MS), with higher percentages of esters identified in vinegars made by IC. The results are encouraging for vinegar production with IC of a mixed A. aceti and K. europaeus culture.

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.

Functional pomegranate beverage production by fermentation with a novel synbiotic L. paracasei biocatalyst.

The recently isolated Lactobacillus paracasei K5 (a potential probiotic) was immobilized on delignified wheat bran (DWB) and was used to produce a functional pomegranate beverage. Fermentations were carried out for 24 h at different pH values (3.3, 3.6, and 3.9), and the fermented juices were stored for 4 weeks at 4 °C. In all cases, the immobilized biocatalyst was shown to be efficient for pomegranate juice fermentation, as indicated by analysis of sugars and organic acids, and ethanol production was maintained at low levels (0.5-1% v/v). The sugars consumed during fermentation were used mainly for cell mass production, while DWB exerted a prebiotic effect, protecting and enhancing the viability of L. paracasei. Optimum viability (9.8-11.7 log cfu/mL) was achieved at pH 3.9. The phenolic content and headspace volatiles were increased after fermentation and storage (112 ±â€¯17-213 ±â€¯30 mg GAE/100 mL and 27-46 compounds, respectively), indicating potential to produce good quality, synbiotic pomegranate beverages.

Revealing new promoters in whey fermentation leads to a new research concept.

The current investigation reveals new findings concerning the substantial promotional activity of orange juice, orange peel and molasses on whey fermentation using single cell culture of kefir microflora. Specifically, the addition in whey of only 1 % v/v orange juice or 1% v/v molasses - 4% w/v orange peel reduced the fermentation time to 10 from 70 hours. But the lowest fermentation time (9 h) was observed when a composite biocatalyst consisted of cells entrapped with orange peel in corn starch gel was used, where also the highest 14C-labeled lactose uptake rate by kefir was recorded. Bio-ethanol concentrations were increased using all the aforementioned promoters too and volatile byproducts such as esters were raised during the whey-orange juice mixtures fermentations, while terpenes by composite biocatalyst. The findings could be the base for new research projects for the rapid production of novel food stuffs and chemicals using whey as raw material.

Production of a Potentially Synbiotic Pomegranate Beverage by Fermentation with Lactobacillus plantarum ATCC 14917 Adsorbed on a Prebiotic Carrier.

A probiotic biocatalyst was prepared through Lactobacillus plantarum ATCC 14917 immobilization on a prebiotic carrier (delignified wheat bran) and was used for fermentations of pomegranate juice. Initially, pomegranate juice was fermented for 24 h and then was stored for 28 days at 4 °C. The obtained results regarding sugar and organic acid analysis revealed that the probiotic biocatalyst was effective. Ethanol was produced in small amounts (0.4-1% v/v). Total phenolic content and antioxidant activity was greater in the fermented pomegranate juice than in unfermented juice after 24 h of fermentation and over the time span of 28 days. Viability of probiotic cells was well maintained (above 8.65 log cfu/mL) after 24 h of fermentation and during 4 weeks of storage at 4 °C, and it is noteworthy that no pathogens were observed. The strength of viability of probiotic cells can be attributed to the immobilization carrier (delignified wheat bran) that exhibits prebiotic properties providing a protective effect to the cells. Finally, the proposed bioprocess of employing the proposed synbiotic biocatalyst for pomegranate juice fermentation shows great potential for commercialization while sensory evaluation highlights the degree of quality of the produced functional pomegranate beverages.

Application of A Novel Potential Probiotic Lactobacillus paracasei Strain Isolated from Kefir Grains in the Production of Feta-Type Cheese.

In the present study 38 lactic acid bacteria strains were isolated from kefir grains and were monitored regarding probiotic properties in a series of established in vitro tests, including resistance to low pH, resistance to pepsin and pancreatin, and tolerance to bile salts, as well as susceptibility against common antibiotics. Among them, the strain SP3 displayed potential probiotic properties. Multiplex PCR analysis indicated that the novel strain belongs to the paracasei species. Likewise, the novel strain (Lactobacillus paracasei SP3) was applied as a starter culture for Feta-type cheese production. Feta-type cheese production resulted in significantly higher acidity; lower pH; reduced counts of coliforms, yeasts and fungi; and improved quality characteristics compared with cheese samples produced with no starter culture. Finally, it is highlighted that the application of the novel strain led to Feta-type cheese production with improved overall quality and sensory characteristics.

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.

Porous cellulose as promoter of oil production by the oleaginous yeast Lipomyces starkeyi using mixed agroindustrial wastes.

Enhanced single cell oil (SCO) production by the oleaginous yeast Lipomyces starkeyi DSM 70296, immobilised on delignified porous cellulose, is reported. Pure glucose media were initially used. The effects of substrate pH and treatment temperature were evaluated, showing that 30°C and pH 5.0 were the optimum conditions for SCO production by the immobilised yeast. The immobilisation technique led to increased lipid accumulation and cell growth by 44% and 8%, respectively, in the glucose media, compared to free cells in suspension. This positive effect was also shown when low concentration mixed agro-industrial waste suspensions were used as substrates, leading to 85% enhanced SCO production in comparison with free cells. Higher fatty acid (HFA) analysis showed that yeast immobilisation led to increased formation of unsaturated HFAs (6%) and reduced saturated HFAs (5%) compared to free cells.

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.

Immobilized rennin in TC/SG composite in cheese production.

The object of the current study was to develop a new process for continuous Feta-type cheese production using a biocatalyst consisting of immobilized rennin on a tubular cellulose/starch gel (TC/SG) composite, which has been proven to be an appropriate carrier for enzyme immobilization. Different methodologies were used in order to prepare four biocatalysts. The most effective was selected for cheese production in a 1L continuous system, providing two economically useful results for the dairy industries: (i) increase of productivity by the continuous coagulation of milk, and (ii) saving of the rennin enzyme expenses of the batch coagulation of milk. The criteria used to choose the appropriate biocatalyst was based on the time of coagulation in successive batches, the concentration of immobilized rennin combined with the filter efficiency and its application in the continuous system. Physicochemical analyses of the cheeses at various stages of the ripening were performed. No significant differences compared to cheeses prepared with the traditional method were found. Aroma compounds were determined by SPME GC-MS.