Optimization of food-grade medium for co-production of bioactive substances by Lactobacillus acidophilus LA-5 for explaining pharmabiotic mechanisms of probiotic.

This study aimed to optimize the co-production of conjugated linoleic acid (CLA), exopolysaccharides (EPSs) and bacteriocins (BACs) by Lactobacillus acidophilus LA-5 in dairy food-grade by-product. The factorial design revealed that the significant factors were temperature, time, and yeast extract. Then the response surface methodology was used for optimization. At the optimal conditions the viable cell number, CLA, EPSs, and inhibition activity were 2.62 ± 0.49 × 108 CFU/mL, 51.46 ± 1.50 µg/mL, 348.24 ± 5.61 mg/mL and 12.46 ± 0.80 mm, respectively. FTIR, GC, TLC, and SDS page analysis revealed the functional groups of pharmabiotics. The FTIR, GC, TLC, and SDS page analysis showed that both CLA isomers (c-9, t-11, and t-10, c-12) produced. The FTIR, GC, TLC, and SDS page analysis indicated that produced EPSs were composed of glucose, mannose, galactose, xylose, and fructose. FTIR, GC, TLC, and SDS page used to report BACs molecular weight, which showed two fractions by molecular mass 35 and 63 kDa. Previously the ability of different probiotic bacteria investigated and optimized the production of CLA, EPSs, and BACs, but, there was no report on the co-producing capacity of these bioactive metabolites by probiotics. The present work was investigated to optimize the co-production of pharmabiotic metabolites by L. acidophilus LA-5, in supplemented cheese whey as a cultivation medium.

Immobilization of ß-galactosidase by halloysite-adsorption and entrapment in a cellulose nanocrystals matrix.

BACKGROUND: Immobilization allows easy recovery and reuse of enzymes in industrial processes. In addition, it may enhance enzyme stability, allowing prolonged use. A simple and novel method of immobilizing ß-galactosidase is reported. Effects of immobilization on the enzyme characteristics are explained. ß-Galactosidase is well established in dairy processing and has emerging applications in novel syntheses. METHODS: ß-Galactosidase was immobilized by physical adsorption on halloysite, an aluminosilicate nanomaterial. Optimal conditions for adsorption were identified. The optimally prepared halloysite-adsorbed enzyme was then entrapped in a porous matrix of nanocrystals of sulfated bacterial cellulose, to further enhance stability. RESULTS: Under optimal conditions, 89.5% of the available protein was adsorbed per mg of halloysite. The most active and stable final immobilized biocatalyst had 1 part by mass of the enzyme-supporting halloysite particles mixed with 2 parts of cellulose nanocrystals. Immobilization raised the optimal pH of the catalyst to 7.5 (from 6.0 for the native enzyme) and temperature to 55 °C (40 °C for the native enzyme). During storage at 25 °C, the immobilized enzyme retained 75.8% of initial activity after 60 days compared to 29.2% retained by the free enzyme. CONCLUSION: The immobilization method developed in this work enhanced enzyme stability during catalysis and storage. Up to 12 cycles of repeated use of the catalyst became feasible. GENERAL SIGNIFICANCE: The simple and rapid immobilization strategy of this work is broadly applicable to enzymes used in diverse bioconversions.

Improvement of lipase biochemical properties via a two-step immobilization method: Adsorption onto silicon dioxide nanoparticles and entrapment in a polyvinyl alcohol/alginate hydrogel.

In this study, the factors affecting lipase adsorption onto SiO2 nanoparticles including SiO2 nanoparticles amounts (8, 19 and 30 mg/mL), lipase concentrations (30, 90 and 150 µg/mL), adsorption temperatures (5, 20 and 35 °C) and adsorption times (1, 12.5 and 24 h) were optimized using central composite design. The optimal conditions were determined as a SiO2 nanoparticles amount of 8.5-14 mg/ml, a lipase concentration of 106-116 µg/mL, an adsorption temperature of 20 °C and an adsorption time of 12.5 h, which resulted in a specific activity and immobilization efficiency of 20,000 (U/g protein) and 60 %, respectively. The lipase adsorbed under optimal conditions (SiO2-lipase) was entrapped in a PVA/Alg hydrogel, successfully. FESEM and FTIR confirmed the two-step method of lipase immobilization. The entrapped SiO2-lipase retained 76.5 % of its initial activity after 30 days of storage at 4 °C while adsorbed and free lipase retained only 43.4 % and 13.7 %, respectively. SiO2-lipase activity decreased to 34.43 % after 10 cycles of use, while the entrapped SiO2-lipase retained about 64.59 % of its initial activity. Compared to free lipase, the Km values increased and decreased for SiO2-lipase and entrapped SiO2-lipase, respectively. Vmax value increased for both SiO2-lipase and entrapped SiO2-lipase.

Saccharomyces cerevisiae and Lactobacillus rhamnosus cell walls immobilized on nano-silica entrapped in alginate as aflatoxin M1 binders.

Aflatoxins are common fungal toxins in foods that cause health problems for humans. The aim of this study was to use Saccharomyces cerevisiae and Lactobacillus rhamnosus cell walls immobilized on nano-silica entrapped in alginate as aflatoxin M1 (AFM1) binders. In this study, microbial walls were disrupted using a three-step mechanical technique including autoclave, thermal shock, and ultrasound. Dynamic light scattering (DLS) results proved size reduction in microbial walls ranging 75.8-91.4 nm. Disrupted walls were immobilized on nano-silica to enhance the efficiency of AFM1 adsorption. Then, to prevent the release of the nano-silica or cell walls into the reaction medium, they were entrapped into alginate gel beads. Fourier transform infrared spectrometer (FT-IR) and scanning electron microscopy (SEM) micrographs confirmed the immobilization and entrapment process. Individual and mixtures of free cell walls, immobilized-entrapped walls, alginate bead and nano-silica were contacted with AFM1 for 15 min and 24 h. AFM1 reduction ability was evaluated using high performance liquid chromatography (HPLC). The results showed an AFM1 reduction ranging 53-87% for free cell walls mixture at 15 min and alginate bead respectively. Also, it was possible to reuse immobilized-entrapped walls as binders with an efficiency of about 85%.

Enhancement of biochemical aspects of lipase adsorbed on halloysite nanotubes and entrapped in a polyvinyl alcohol/alginate hydrogel: strategies to reuse the most stable lipase.

Entrapment of halloysite nanotubes (HNTs) loaded with enzyme, into a polymer matrix (PVA/Alg), is a way to produce an environment surrounding the adsorbed enzyme molecules which improves the enzyme properties such as storage and operational stability. Hence, in this study, we optimised the factors affecting lipase adsorption onto halloysite nanotubes including halloysite amounts (5, 42.5 and 80 mg), lipase concentrations (30, 90 and 150 µg/ml), temperatures (5, 20 and 35 °C) and adsorption times (30, 165 and 300 min). The optimal conditions were determined as an halloysite amount of 50 to 80 mg, a lipase concentration of 30 to 57 µg/ml, an adsorption temperature of 20 °C and an adsorption time of 165 min, which resulted in a specific activity and adsorption efficiency of 15,000 (U/g protein) and 70%, respectively. Then, lipase adsorbed under optimal conditions was entrapped in a PVA/Alg hydrogel. The formation mechanism of immobilized lipase was investigated by FESEM and FTIR. Subsequent entrapment of adsorbed lipase improved the lipase storage and operational stability. Km, Vmax, Kcat and Kcat/Km values showed an increase in the entrapped HNT-lipase performance in comparison with the free and adsorbed lipase.

Development and characterization of biocomposite films made from kefiran, carboxymethyl cellulose and Satureja Khuzestanica essential oil.

Kefiran-carboxymethyl cellulose biocomposite films incorporated with Satureja Khuzestanica essential oil were developed and characterized. Results indicated that increase in the concentration of the essential oil increased ultimate tensile strength and contact angle but decreased elongation at break, moisture content and water vapor permeability. It also significantly altered color parameters and the percentage of light transmission in the visible and ultraviolet range. Fourier transform infrared spectroscopy revealed the formation of hydrogen bonds between polymer matrix and essential oil. Scanning electron microscopy showed that the surface structure of the films was homogeneous without porosity. Increase in storage modulus and glass transmission temperature in films incorporated with the essential oil was observed through dynamic mechanical thermal analysis. Moreover, significant increase in antioxidant properties and phenolic compounds were noticed. Ultimately, results obtained from evaluation of antimicrobial characteristics of films indicated their inhibitory effects against Staphylococcus aureus and Escherichia coli bacteria.

Immobilization of α-amylase on chitosan-montmorillonite nanocomposite beads.

Enzyme immobilization is a way to increase efficiency of the enzyme and facilitate its recovery. The aim of this study was to immobilize α-amylase on chitosan-montmorillonite nanocomposite beads. Nanocomposite beads were prepared as the carrier for the enzyme stabilization and their surface was modified by Glutaraldehyde. Alpha-amylase was immobilized on nanocomposite beads by covalent bonding. The results of scanning electron microscopy (SEM) showed that particle size range of montmorillonite was 10-30 nm. This study indicated that the enzyme immobilization efficiency was 87%. The activity of free and immobilized enzyme during 40 days of storage at 4 °C decreased 95% and 36%, respectively. The results showed that the immobilized enzyme activity after reusing five times decreased about 47%. This study indicated that the immobilized enzyme activity was higher than the free enzyme at different temperatures. Also the immobilized enzyme was more stable than the free enzyme at lower pH. The results of kinetic parameters showed that Km values of the immobilized enzyme (9.12 µmol/ml) were higher than free enzyme (6.80 µmol/ml). The Vmax values for the free and immobilized enzyme were 1.30 and 0.629 µmol/mg·min, respectively.

Preparation and characterization of active emulsified films based on chitosan-carboxymethyl cellulose containing zinc oxide nano particles.

Active nanocomposites based on carboxymethyl cellulose-chitosan-oleic acid (CMC-CH-OL) incorporated with different concentrations (0.5-2wt.%) of zinc oxide nanoparticles (ZnO NPs) were produced by casting method. The effects of ZnO NPs on the morphological, mechanical, thermal, physical and antifungal properties of the films were studied. New interaction between ZnO NPs and polymer matrix were confirmed by Fourier Transform infrared. After addition of ZnO NPs, tensile strength, lightness (L*) and thermal stability decreased however, elongation at break, contact angle, a* (greenness) and b* (yellowness) of the nanocomposite films increased in comparison to the films without nano-filler. UV transmittance at 280nm decreased from 17.3% to 0.2, 0.1 and 0.1 for the nanocomposite films containing 0.5, 1 and 2wt.% ZnO NPs, respectively, suggesting higher UV blocking properties. Disc diffusion test showed considerable antifungal properties of the active nanocomposite films against Aspergillus niger, especially in CMC-CH-OL-ZnO 2wt.% by more than 40% fungal growth inhibition.

Lactobacillus plantarum as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity.

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.

Synbiotic yogurt-ice cream produced via incorporation of microencapsulated lactobacillus acidophilus (la-5) and fructooligosaccharide.

Yogurt-ice cream is a nutritious product with a refreshing taste and durability profoundly longer than that of yogurt. The probiotic Lactobacillus acidophilus (La-5) cells either in free or encapsulated form were incorporated into yog-ice cream and their survivability were studied. Fructooligosaccharide (FOS) as a prebiotic compound at three levels (0, 4 & 8 % w/w) was added to yogurt-ice cream mix and its effects on some chemical properties, overrun and firmness of product were evaluated. The higher the incorporated FOS concentration, the lower were the pH value and higher the total solid content of treatments. FOS incorporation (8 %) significantly increased the overrun of treatments and reduced their firmness. The viable counts of free probiotics decreased from ~9.55 to ~7.3 log cfu/g after 60 days of frozen storage while that of encapsulated cells merely decreased less than 1 log cycle. Encapsulation with alginate microbeads protected the probiotic cells against injuries in the freezing stage as well as, during frozen storage.