Clarification Of Orange Juice Research Articles

Purification and biochemical characterization of a novel thermostable endo-polygalacturonase from Aspergillus niger strain HO32 and its suitability for clarification of orange juice

A novel endo-polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH2-terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na+, Ca2+, Mg2+, Mn2+, and Zn2+) positively affect PGC-AN64 activity, while Ba2+, Cd2+, and Cr3+ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification.

Effect of Support Matrix and Crosslinking Agents on Nutritional Properties of Orange Juice during Enzyme Clarification: A Comparative Study.

This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, including reducing sugars, total soluble solids, vitamin C, and phenolic compounds, juice color, and pH, were evaluated. The results revealed that the immobilization on chitosan activated using genipin resulted in the highest biocatalyst activity (1211.21 U·g-1). The juice treatments using the biocatalysts led to turbidity reduction in the juice (up to 90%), with the highest reductions observed in treatments involving immobilized enzyme on chitosan. Importantly, the enzymatic treatments preserved the natural sugar content, total soluble solids, and pH of the juice. Color differences between treated and raw juice samples were especially relevant for those treated using enzymes, with significant differences in L* and b*, showing loss of yellow vivid color. Analysis of phenolic compounds and vitamin C showed no significant alterations after the enzymatic treatment of the raw juice. According to our results, the clarification of orange juice using immobilized enzymes can be a compromise in turbidity reduction and color reduction to maintain juice quality.

Saccharification of Agricultural Wastes and Clarification of Orange Juice by Penicillium rolfsii CCMB 714 Pectinase

Pectinases are enzymes used in several industrial processes. Seven agroindustrial wastes—jackfruit seed meal (Artocarpus heterophyllus), cocoa seed peel (Theobroma cacao), cocoa husks (Theobroma cacao), passion fruit husks (Passiflora edulis), mangosteen husks (Garcinia mangostana), malt residue (Hordeum vulgare) and the peach palm waste (Bactris gasipaes Kunth.)—were evaluated to produce a crude extract containing pectinase activity by Penicillium rolfsii CCMB 714. The jackfruit seed meal was chosen as the best substrate for solid-state fermentation, which was optimized with 4 mL of water as a wetting agent for 2 days at 35 °C and with a 0.5% nitrogen source, whereby the pectinase production increased by 44% (362.09 U/g). The obtained crude extract was characterized and applied to wastes saccharification and orange juice clarification. The pectinase showed better activity at a pH of 3.0 to 5.0 and 55 °C, it stably maintained over 80% of activity at 30–50 °C for up to 60 min and 1 mM CuSO4 increased the pectinase activity by 17%. The saccharification of agroindustrial wastes (cocoa husks, mangosteen husks and passion fruit husks) resulted in 126.55 µmol/mL of reducing sugars from passion fruit husks, which represents an increase of 126% after optimization (45 °C for 22 h). For the clarification of orange juice, it was possible to reduce the absorbance of the juice by 55%. These results elucidate the potential of the low-cost pectinase solution from P. rolfsii CCMB 714 cultivated in jackfruit seed meal for both the enzymatic pretreatment of plant biomass and the application in beverage industries.

Low-Fouling Plate-and-Frame Ultrafiltration for Juice Clarification: Part 2—Module Design and Application

The purification and concentration of orange juice are crucial to remove undesirable materials, such as pectin, which is responsible for juice clouds; or limonene, which is responsible for bitter taste. Membrane-based juice clarification is preferred due to its capability to separate specific targeted molecules, while still maintaining the clarified juice’s nutritional content. In this study, a novel designed bench-scale plate-and-frame membrane module composed of low fouling cellulose acetate membrane sheets was manufactured to facilitate orange juice clarification. The experimental results demonstrated the effectiveness of the developed module to be used for juice clarification. After incorporating the functional and structural design parameters, the final module had the following specifications: dimensions of 125 × 168 mm, an effective volume of 0.9–9.4 L, a total active membrane area of 1088 cm2, and a transmembrane pressure of 0.3–0.55 MPa. The results of the juice clarification show no difference in the value of pH, viscosity, total acid, water content, color L* (brightness), and color a* (reddish) of the feed, the permeate, and the retentate streams. The clarified juice had slightly higher total dissolved solids (°Brix), ash content, vitamin C, and color (b* yellowish). Overall, our findings demonstrated that the developed plate-and-frame module could effectively be used to clarify orange juice without altering the quality, i.e., reducing the nutritional contents.

FRUIT JUICES CLARIFICATION WITH  AMYLASES

On the market consumers prefer clear fruits juice. The clarification process can be improved by utilization of pectolytic enzymes with major influence in juice chemical composition. This study follows the possibility to use in apple, pear and orange juice clarification process with untypical enzymes for industrial processes – amylases.

Immobilization of xylanase on differently functionalized silica gel supports for orange juice clarification

The immobilization of xylanase from Trichoderma longibrachiatum via ion exchange on 3-propylsulfonic acid (Xyl/Si-SO3) and 3-aminopropyl (Xyl/Si-NH3) functionalized silica gel and covalently on 3-carboxypropyl (Xyl/Si−COOH) and silica gel functionalized with 3-aminopropyl and modified with glutaraldehyde (Xyl/Si-Glu) were performed and the prepared biocatalysts were used for clarification of orange juice. The supports and immobilized biocatalysts were characterized by scanning electron microscopy, and thermal gravimetric analysis. Free and all immobilized xylanase samples showed maximal catalytic activity at pH 6.0. The free xylanase and Xyl/Si-NH3 had a maximal catalytic activity at 50 °C, while Xyl/Si-Glu, Xyl/Si−COOH and Xyl/Si-SO3 exhibited their maximal catalytic activities at 60 °C. The Xyl/Si-Glu showed 4.2-fold higher thermal stability than the free xylanase at 50 °C and pH 6.0. The clarity of orange juice was increased by 74.7, 73.6, 59.3, 60.3, and 78.8 % respectively by using free xylanase, Xyl/Si-Glu, Xyl/Si−COOH, Xyl/Si-SO3 and Xyl/Si-NH3 at 50 °C after 180 min treatment time. The reusability studies showed that Xyl/Si-Glu, Xyl/Si−COOH, Xyl/Si-SO3 and Xyl/Si-NH3 retained 77, 71, 11 and 16 %, respectively, of their initial activities after 10 reuses. These results show that Xyl/Si-Glu and Xyl/Si−COOH are better potential candidates than Xyl/Si-SO3 and Xyl/Si-NH3 in fruit juice clarifying processes.

Statistical Optimization of Media Components for Xylanase Production by Aspergillus spp. Using Solid State Fermentation and its Application in Fruit Juice Clarification

Xylanases are enzymes that convert xylan into xylose, xylobiose, and xylotriose. The present study deals with the production and optimization of xylanase through Solid-State Fermentation (SSF) using different agricultural wastes by Aspergillus spp. The Plackett Burman (PB) design was used to screen significant media components affecting the xylanase production. The carbon sources screened were wheat bran, rice bran, sugarcane bagasse, corn cob, and orange peel. The nitrogen sources screened were yeast extract, peptone, (NH4)2SO4, Na2NO3, and urea. Also, nine different salts such as KCl, MgSO4, Na2HPO4, CaCl2, FeSO4, ZnSO4, Na2CO3, KH2PO4, and NaH2PO4 which act as trace elements were screened. The results showed that wheat bran, yeast extract, Na2NO3 and KCl are the significant factors that affect xylanase production. A 33 Full Factorial Design (FFD) was performed to optimize the significant media components (wheat bran, KCl, yeast extract) obtained from PB design using Response Surface Methodology (RSM). Statistical analysis of results showed that wheat bran, KCl, yeast extract, and interaction between wheat bran and yeast extract were found to be significant. The optimum concentration of wheat bran, KCl, yeast extract was 8 g/L, 0.1 g/L and 3 g/L. The Partial purification of xylanase was carried out using ammonium salt precipitation and dialysis. Gel filtration chromatography was performed to optimize the elution time, which was found to be 6 minutes. Application of xylanase in orange juice clarification was studied at 40 °C, 50 °C, and 60 °C. The optimum temperature obtained was 60 ºC.

Co-culturing corncob-immobilized yeasts on orange peels for the production of pectinase.

Pectinase is an industrially important enzyme which is employed in an array of commercial processes; cost of production, however, impedes its application. The main objective of this study was to design a two-layered strategy for the reduction of production cost, firstly by using a yeast co-culture in an immobilized form on an agricultural waste matrix, corncob (CB), secondly by utilizing orange peels (OP) as substrate. Two yeast strains, Saccaromyces cerevisiae MK-157 and Geotrichum candidum AA15 were cultivated as mono-, as well as, co-culture after immobilization on CB and pectinase production was monitored. Initial experiments revealed that co-culture is beneficial to get sustainable product in subsequent 2nd and 3rd production cycles. The factors affecting pectinase production in consecutive three production cycles were studied by employing Plackett-Burman design and the significant factors were optimized through Box-Behnken design. Under optimized conditions, 17.89IUmL-1 of pectinase was obtained. Scanning electron micrographs presented damaged immobilized yeast cells on CB after the 3rd production cycle. The pectinase production was improved substantially by using immobilized co-culture and hence the strategy was found effective at lab scale. Since, pectinase is applied in orange juice clarification, therefore, the study can be extended to move forward towards circular economy.

Highly alkali-stable and cellulase-free xylanases from Fusarium sp. 21 and their application in clarification of orange juice

Xylanase is a versatile tool in the food, fiber biobleaching and biofuel industries. Here, to discover new enzyme with special properties, we cloned three xylanases (Xyn11A, Xyn11B, and Xyn11C) by mining the genome of the xylanase producing fungus strain Fusarium sp. 21, biochemically characterized these enzyme and explored their potential application in juice processing. Both Xyn11A and Xyn11B had an optimal pH of 6.0 and optimal temperature of 45 °C, and retained >90% of the residual activity at pH range of 5–10.5 for 24 h. Xyn11C displayed the maximum activity at pH 5.0 and 45 °C and outstanding pH stability with a minimal loss of activity in the pH range of 2.0–10.5. These three xylanases displayed a strong specificity towards beechwood and corncob xylan, with no activity for other substrates. Xyn11A showed much a higher activity against corncob xylan, while Xyn11B and Xyn11C presented higher activities against beechwood xylan. Xyn11A catalyzed the hydrolysis of beechwood xylan with a Km of 4.25 ± 0.29 mg·mL−1 and kcat/Km of 30.34 ± 0.65 mL·s−1·mg−1, while the hydrolysis of corncob xylan had Km and kcat/Km values of 14.73 ± 1.43 mg·mL−1and 26.48 ± 0.11 mL·s−1·mg−1, respectively. Xyn11B and Xyn11C hydrolyzed beechwood xylan with Km of 9.8 ± 0.69 mg·mL−1, and 4.89 ± 0.38 mg·mL−1and kcat/Km values of 45.07 ± 1.66 mL−1·mg−1, and 26.95 ± 0.67 mL·s−1·mg−1, respectively. Beechwood xylan hydrolysates catalyzed by these three xylanases contained xylobiose, xylotriose and xylooligosaccharides (XOS). The clarification of orange juice was improved when treated with these three xylanases. Conclusively, the desirable pH stability and substrate specificity make Xyn11A, Xyn11B and Xyn11C have high potential for application in fiber biobleaching, wine and fruit juice clarification, as well as probiotic XOS production.

Enzymatic clarification of orange juice in continuous bed reactors: Fluidized-bed versus packed-bed reactor

In this study, a commercial enzyme cocktail designed for fruit juice clarification was immobilized on chitosan beads activated with glutaraldehyde and used for setting-up continuous packed-bed and fluidized-bed reactors. Firstly, the enzyme cocktail concentration in the immobilization process was tested to get maximum biocatalyst activity in the reaction. Then, the effect of pH and temperature on the free and immobilized enzyme cocktail stability and activity were studied. Despite the low enzyme cocktail activity recovery after immobilization (1 %), the stability of immobilized enzyme was greater compared to free enzyme, even under extreme reaction conditions. For example, when activity was measured at 90 °C and pH 4.8, the immobilized enzyme cocktail retained 80 % of its initial activity while the free enzyme retained only 35 %. Finally, the clarification capacity of the biocatalysts using orange juice was tested, providing clearer juices when performed in the fluidized-bed reactor, showing 60 % of its initial clarification capacity after 72 h of continuous use. Otherwise, the clarification rate in the packed-bed reactor decreased linearly during 54 h, probably due to the formation of dead zones and preferential paths along the bed. Pectinase immobilized on chitosan particles and used in fluidized-bed reactor seems to be a good alternative for large scale application on juice clarification.

Citrus limetta peels: a promising substrate for the production of multienzyme preparation from a yeast consortium

Citrus limetta peels (CLP), a waste material generated by juice industries, has scarcely been reported for the production of yeast enzymes. The study was conducted to obtain a multienzyme preparation from a yeast consortium under solid-state fermentation of CLP. The substrate, CLP, was pretreated using either acid or alkali, and factors affecting production of multienzyme were studied by generating two separate Plackett–Burman designs. Since, alkali-pretreated CLP yielded higher titers; therefore, significant factors affecting multienzyme preparation using this substrate were optimized by employing Box–Behnken design. The analysis revealed that under optimized conditions, i.e., cultivation of yeast strains for 72 h to alkali-pretreated CLP moistened with mineral salt medium having pH 5 yielded more than 10 IU mL−1 of cellulase, xylanase, and amylase. The multienzyme was studied for its application to saccharify fruit and non-fruit wastes and for orange juice clarification. The data showed that the enzyme preparation could release 3.03 mg L−1 h−1 of reducing sugars from various crude substrates and was able to reduce turbidity of orange juice by 11% with substantial decrease in viscosity and acidity. Hence, CLP appeared as a promising substrate to produce multienzyme preparation from yeast consortium.

Immobilization of pectinase on chitosan-magnetic particles: Influence of particle preparation protocol on enzyme properties for fruit juice clarification

Magnetic-chitosan particles were prepared following three different protocols enabling the preparation of particles with different sizes – nano (Nano-CMag, Micro (Micro-CMag) and Macro (Macro-CMag) – and used for pectinase immobilization and clarification of grape, apple and orange juices. The particle size had a great effect in the kinetic parameters, Nano-CMag biocatalyst presented the highest Vmax value (78.95 mg. min−1), followed by Micro-CMag and Macro-CMag, with Vmax of 57.20 mg.min−1 and 46.03 mg.min−1, respectively. However, the highest thermal stability was achieved using Macro-CMag, that was 8 and 3-times more stable than Nano-CMag and Micro-CMag biocatalysts, respectively. Pectinase immobilized on Macro-CMag kept 85% of its initial activity after 25 batch cycles in orange juice clarification. These results suggested that the chitosan magnetic biocatalysts presented great potential application as clarifying catalysts for the fruit juice industry and the great importance of the chitosan particles preparation on the final biocatalyst properties.

Characterization of pectinase from Geotrichum candidum AA15 and its potential application in orange juice clarification

PurposePectinase is used in fruit juice industry to increase the juice yield and to enhance clarification process by reducing turbidity and viscosity. The current study was focused on characterizing pectinase preparation from Geotrichum candidum AA15 and to evaluate its application in clarification of orange juice. MethodsThe pectinase preparation from AA15 was characterized to determine optimum temperature and pH for its activity and thermal stability under acidic environment. Considering the desirable attributes exhibited by the enzyme, it was applied for orange juice clarification and factors affecting the clarification process were optimized using response surface methodology (RSM) approach. ResultsThis enzyme exhibited the highest activity when incubated at 35 °C for 25 min in a buffer of pH 5. The enzyme did not lose any activity for up to 1 h at 30 °C. Whereas, activity was declined to 82% and 67% when incubated for 1 h at 35 °C and 40 °C, respectively. Moreover, stability of the enzyme when placed in the buffer of pH 2–3 was remarkable. The characteristics of extracted pectinase to work at mesophilic temperature under acidic environment presented it as suitable to be applied in fruit juice processing industries. Consequently, the factors affecting orange juice clarification, including temperature, treatment duration and enzyme loadings were optimized using RSM approach and clarification yield of 61% was obtained under optimized conditions. ConclusionThe pectinase preparation was found suitable to clarify orange juice and the data depicted the possible candidature of the strain for future biotechnological applications.

Screening and Characterization of Polygalacturonase as Potential Enzyme for Keprok Garut Orange (Citrus nobilis var. chrysocarpa) Juice Clarification

Use of thermostable enzyme from bacilli for industrial application is significant. This research aimed to isolate thermophilic pectinolytic bacteria from orange peel and vegetable waste which produced thermostable polygalacturonase, to investigate the polygalacturonase ability in clarifying keprok Garut orange juice, and to characterize polygalacturonase based on pH optimum, temperature optimum, enzyme stability, enzyme kinetics KM, and Vmax. Obtained, 14 isolates that further selected to 4 best isolates based on highest polygalacturonase activity and keprok Garut orange juice clarification ability. Four selected enzyme isolates were AR 2, AR 4, KK 4, and KK 5 had ability to increase juice transmittance, decrease juice viscosity and also reduce total soluble solid. Furthermore 4 selected isolates were partially purified by ammonium sulphate precipitation and dialysis method. Four partially purified enzymes were known that enzyme character of AR 2 optimum at pH 6; AR 4 optimum at pH 5.5; KK 4 optimum at pH 6; and KK 5 optimum at pH 4.5. Four enzymes were optimum at temperature 60°C thus stable at temperature 50-60°C, this characteristic indicate that enzymes were thermostable. AR 2 showed active activity stable at pH 4-7; AR 4 showed active activity stable at pH 6-7; KK 4 showed active activity stable at pH 4-6; however KK 5 stable at pH 4-5. Enzyme AR 2 and KK 4 was getting inactive at pH 11, thus AR 4 and KK 5 inactive at pH 12. KM value of AR 2, AR 4, KK 4, and KK 5 was 0.0959; 0.0974; 0.0966; and 0.178 mg/ml respectively. Vmax of AR 2, AR 4, KK 4, and KK 5 was 0.0203; 0.0202; 0.0185; and 0.0229 U/ml respectively. Enzyme AR 2 was the most compatible enzyme to be applied in keprok Garut orange juice clarification for it had the lowest KM value.

Characterization of biosynthesized chitosan nanoparticles from Penaeus vannamei for the immobilization of P. vannamei protease: An eco-friendly nanobiocatalyst

ABSTRACTNanomaterials are being applied due to their excellent characteristics, such as the high surface area-to-volume ratio, excellent physicochemical properties, and biological compatibility. Chitosan is an amino polysaccharide and a versatile, second-most abundant natural polymer. In this presented work, chitosan was purified from Penaeus vannamei from the Persian Gulf and the homogeneity level was used a combination of three procedures: deproteinization, demineralization, and deacetylation. Chitosan nanoparticles were synthesized by the ionization gelation technique. P. vannamei protease immobilized on the surface of the positively charged chitosan nanoparticles through ionic or electrostatic interaction. The immobilized enzyme exhibited better tolerance to variations in the medium pH and temperature, enhanced pH and temperature stability, improved storage stability, and good reusability compared with the free enzyme. The Michaelis–Menten kinetic constant (Km), the maximum reaction velocity (Vmax), and the catalytic efficiency (kcat) for native P. vannamei protease were 2.5 µM, 87 µM. min−1, and 132 min−1, respectively, whereas for immobilized enzyme, Km, Vmax, and kcat values were 2.7 µM, 83 µM. min−1, and 128 min−1, respectively. The effect of both soluble and immobilized P. vannamei protease on the clarification of orange juice was also analysed.

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Polygalacturonases (PG) represent an important member of pectinases group of enzymes with immense industrial applications. A fungal strain Aspergillus niger MTCC478 was used for the production of polygalacturonase both under submerged and solid-state fermentation condition. Further its production was optimized under solid-state fermentation condition with media comprising of wheat bran and tea extract. Purification of an exo-PG was achieved by acetone precipitation (60–90%) and CM-cellulose column chromatography revealing 15.28-fold purification with a specific activity of 33.47 U/mg protein and 1.2% yield. A relative molecular mass of purified PG was approximately 124.0 kDa. The pH and temperature optimum was found to be 4 and 50 °C, respectively. The kcat and Km value for degradation of PGA by the purified enzyme was found to be 194 s−1 and 2.3 mg/mL, respectively. Cu2+ was found to enhance the PG activity while Ag+ completely inhibited the enzyme activity. The application of the purified PG in orange juice clarification was elucidated.

Valorization of Corn-Cob by Fungal Isolates for Production of Xylanase in Submerged and Solid State Fermentation Media and Potential Biotechnological Applications

The increasing awareness of importance of xylanases in various industrial processes has led to intensive research in effective production of xylanases. In this study, eight fungal strains were screened for the production of xylanases in submerged (SmF) and solid state fermentations (SSF) in corncob-based media. Positive results of reddish orange halo-zones of hydrolysis were obtained for qualitative screening. In SmF, xylanase activity from Aspergillus fumigatus SD5A yielded highest activity (50.55 U/ml) in 168 h, while Aspergillus flavus SD4A, A. fumigatus L1, Fusarium solani SD3C, Aspergillus niger L3, Trichoderma longibrachiatum L2, Botryodiplodia sp. L5 and A. flavus L4, showed xylanase activities ranging from 10.38 to 44.81 U/ml. In SSF, xylanase activities ranged from 12.30 to 48.63 U/g in 120 h, with the highest activity obtained from A. fumigatus L1. Optimum temperatures for xylanases obtained in SmF ranged from 55 to 70 °C, while that of SSF ranged from 50 to 70 °C. Also, the optimum pH for xylanases obtained in both SmF and SSF ranged from 5.0 to 7.0. All the fungi did not produce aflatoxin on neutral red desiccated coconut agar. The fungal xylanases improved dough rising of bread and clarification of orange juice by 1.87–2.2-folds and 58.12–74.22% respectively. The results reported here showed that the range of characteristics exhibited by the xylanases could make them widely applicable in various industries, and also an important way for the valorization of agricultural waste.

Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 7589

Abstract Polygalacturonases represents an important member of pectin-degrading glycoside hydrolases of the family GH28 and is widely distributed among fungi, bacteria, yeasts, plants and some plant parasitic nematodes. Microbial polygalacturonases are widely used in food industry to clarify fruit and vegetable juice by degrading pectin. An exo- polygalacturonase from Aspergillus flavus MTCC 7589 was purified, biochemically characterized and its utility in clarification of orange juice was elucidated. The enzyme was purified by acetone precipitation (0–60%), CM- cellulose column chromatography and Gel filtration chromatography using Sephadex G-100 resulting in 8.24 fold purification with specific activity of 26.8 IU/mg protein and yield of 0.9%. A single band corresponding to a relative molecular mass of approximately 29.0 kDa was revealed by SDS-PAGE. The enzyme showed pH and temperature optima of 5 and 40 °C respectively. The K m and kcat values of purified enzyme were 2.08 mg/mL and 96 s − 1 respectively. The metal ions Co 2+ , Cu 2+ and K + were found to stimulate PG activity while Ca 2+ , Ag + and Hg 2+ ions were inhibitory. The preliminary study revealed its potential in clarification of orange juice.

The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis

Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4°C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22μm-filtered OJ at 24h post-inoculation, compared to filtered OJ samples containing particles between 0.22μm and 11μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7μm-filtered OJ and both 0.22μm-filtered and 1.2μm-filtered OJ after 24hour incubation at 22.5°C. This indicated that OJ cloud between 0.7μm and 0.22μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4°C, while the FCM viable count did not substantially decrease until 48h, decreases in TVC were observed between 0 and 48hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in monitoring bacterial physiology in foods, and potential effects of OJ clarification on bacterial physiology.

Development of an original lab-scale filtration strategy for the prediction of microfiltration performance: Application to orange juice clarification

Abstract In food industries, microfiltration is widely used for the separation of particles and/or microorganisms in many beverages as fruit juices. However, like in many other applications, an accurate prediction of juices filterability is not yet validated; filtration tests in lab-scale dead-end filtration units have been challenged by frequent deficiencies to describe filtration performance at large-scale. The aim of this work was to develop a suitable lab-scale filtration strategy in order to anticipate membrane fouling during crossflow microfiltration of orange juices, through a relevant evaluation of their fouling behavior. The impact of membrane average pore size, juice particles size distribution and shear forces on filtration tests in dead-end filtration cell was explored through a D-optimal experimental design. Microfiltration experiments at pilot-scale were carried out in order to demonstrate the relevance of the proposed strategy. Results showed that an anticipation of juice fouling propensities, at pilot-scale, was possible when stirred dead-end filtration test was carried out under the following conditions: ceramic membrane with an average pore size of 0.2 μm, juice fractionation prior to filtration (500 g/1 min) and rotational speed of the blade of 1000 rpm. These conditions, notably the ones related to the particle back-transport and fouling mechanisms, showed that dead-end filtration cell can be used as a process-specific anticipation tool to predict membrane fouling during fruit juices pilot-scale microfiltration.