Identification of Cellulose-Degrading Bacteria and Assessment of Their Potential Value for the Production of Bioethanol from Coconut Oil Cake Waste.

Bioconversion of lignocellulosic biomass is a highly promising alternative to rapidly reduce reliance on fossil fuels and greenhouse gas emissions. However, the use of lignocellulosic biomass is limited by the challenges of efficient degradation strategies. Given this need, Bacillus tropicus (B. tropicus) with cellulose degradation ability was isolated and screened from rotten dahlia. The strain efficiently utilized coconut oil cake (COC) to secrete 167.3 U/mL of cellulase activity. Electron microscopy results showed significant changes in the structure and properties of cellulose after treatment with B. tropicus, which increased the surface accessibility and the efficiency of the hydrolysis process. The functional group modification observed by Fourier transform infrared spectroscopy indicated the successful depolymerization of COC. The X-ray diffraction pattern showed that the crystallinity index increased from 44.8% to 48.2% due to the hydrolysis of the amorphous region in COC. The results of colorimetry also reveal an efficient hydrolysis process. A co-culture of B. tropicus and Saccharomyces cerevisiae was used to produce ethanol from COC waste, and the maximum ethanol yield was 4.2 g/L. The results of this work show that B. tropicus can be used to prepare biotechnology value-added products such as biofuels from lignocellulosic biomass, suggesting promising utility in biotechnology applications.

Influence of the Emulsifier Sodium Caseinate-Xanthan Gum Complex on Emulsions: Stability and Digestive Properties.

In this study, virgin coconut oil (VCO) nanoemulsions were prepared by ultrasonication using a sodium caseinate (SC) and xanthan gum (XG) complex as an emulsifier. The stability and digestion characteristics of SC/XG-VCO emulsions formed by co-adsorption and SC-VCO-XG emulsions formed by layer adsorption were compared. The stability of the two emulsions was studied under different pH, ionic strength, heat treatment, freeze-thaw cycles, and storage conditions, and the droplet size and zeta potential were used as indicators to assess the stability. In addition, the stability of oxidation and the digestive properties of both emulsions were studied. It was found that the SC-VCO-XG emulsions had better environmental stability, oxidative stability, storage stability, and digestibility compared to SC/XG-VCO emulsions. This study has shown that the formation method of protein-polysaccharide stabilized emulsions has an impact on the stability and digestibility properties of the emulsions, and that the emulsion carriers constructed by layer adsorption are more suitable for subsequent industrial production and development.

Nanosized niosomes as effective delivery device to improve the stability and bioaccessibility of goat milk whey protein peptide.

This study sought to develop a nanoscale delivery system to enhance the stability and bioaccessibility of goat milk whey protein peptides. Goat milk whey protein was hydrolyzed by papain, and the hydrolysate was ultrafiltered to obtain a low molecular weight peptide (GWP, <3 kDa) with strong hypoglycemic activity. The GWP-loaded liposomes and niosomes encapsulation systems were prepared using phytosterols (ergosterol, ß-sitosterol, mixed phytosterols, and stigmasterol) instead of cholesterol. Results showed that the GWP-loaded niosomes (GWP-NS) prepared from ß-sitosterol had the higher GWP encapsulation efficiency (90.46 ± 4.02 %) and the smaller particle size (92.07 ± 9.8 nm) than liposomes (GWP-LS). Additionally, the morphological results showed that two GWP-loaded systems were smooth and spherical, and the FT-IR spectroscopy confirmed the successful formation of the peptide-loaded system. Compared with GWP, GWP-LS and GWP-NS showed the higher stability under different pH, temperature, and NaCl concentration conditions, especially GWP-NS. Furthermore, GWP-NS could significantly improve the retention rate of GWP during simulated gastrointestinal digestion, in vitro bioaccessibility, and hypoglycemic activity. These findings suggest that ß-sitosterol could be a potential membrane stabilizer alternative to cholesterol, and GWP niosomes could be a potential new drug delivery system.

Screening of cellulose-degrading yeast and evaluation of its potential for degradation of coconut oil cake.

Coconut oil cake (COC), a byproduct of oil extraction, contains high levels of cellulose. The aim of this study was to isolate a cellulose-degrading yeast from rotten dahlia that can effectively use COC as the only carbon source for cellulase secretion. Based on screening, Meyerozyma guillermondii CBS 2030 (M. guillermondii) was identified as a potential candidate, with the highest cellulolytic activity among the yeast strains isolated, with the carboxymethyl cellulase (CMCase) activity reaching 102.96 U/mL on day 5. The cellulose in COC samples was evaluated before and after degradation by M. guillermondii. Analysis based on field emission scanning electron microscopy (FESEM) revealed that the COC structure was changed significantly during the treatment, indicating effective hydrolysis. Fourier transform infrared spectroscopy (FTIR) of the modified functional groups indicated successful depolymerization of coconut cake. X-ray diffraction (XRD) and analysis of color differences established effective degradation of COC by M. guillermondii. The results demonstrate that M. guillermondii effectively secretes CMCase and degrades cellulose, which has important practical significance in COC degradation.

Effects of Polysaccharide Concentrations on the Formation and Physical Properties of Emulsion-Templated Oleogels.

An emulsion template method was an effective way to prepare oleogels. However, there were few reports on how hydroxypropyl methylcellulose-pectin (HPMC-PC) mixtures affected the physicochemical properties of the obtained oleogels. In this study, the oleogels were prepared by an emulsion template method. The influences of HPMC and PC concentrations on the formation and physical properties of the emulsions and oleogels were investigated, by analyzing particle size distribution, microstructure, rheological test, oil loss, and crystallinity. The results of particle sizes and microstructure showed that a high concentration of HPMC and PC exhibited a better emulsification performance. The rheological tests indicated that a high concentration of HPMC and PC contributed to an increase in the mechanical strength of emulsions and oleogels. Moreover, an increase in an HPMC and PC concentration was beneficial to reduce the oil loss of oleogels. However, the change of HPMC and PC concentrations had no significant effect on the X-ray diffraction pattern of oleogels. This study could provide a theoretical basis for the construction of polysaccharide-based oleogels.

Pathogen-Specific Bactericidal Method Mediated by Conjugative Delivery of CRISPR-Cas13a Targeting Bacterial Endogenous Transcripts.

The emergence of antibiotic-resistant bacteria threatens public health, and the use of broad-spectrum antibiotics often leads to unintended consequences, including disturbing the beneficial gut microbiota and resulting in secondary diseases. Therefore, developing a novel strategy that specifically kills pathogens without affecting the residential microbiota is desirable and urgently needed. Here, we report the development of a precise bactericidal system by taking advantage of CRISPR-Cas13a targeting endogenous transcripts of Salmonella enterica serovar Typhimurium delivered through a conjugative vehicle. In vitro, the CRISPR-Cas13a system exhibited specific killing, growth inhibition, and clearance of S. Typhimurium in mixed microbial flora. In a mouse infection model, the CRISPR-Cas13a system, when delivered by a donor Escherichia coli strain, significantly reduced S. Typhimurium colonization in the intestinal tract. Overall, the results demonstrate the feasibility and efficacy of the designed CRISPR-Cas13a system in selective killing of pathogens and broaden the utility of conjugation-based delivery of bactericidal approaches. IMPORTANCE Antibiotics with broad-spectrum activities are known to disturb both pathogens and beneficial gut microbiota and cause many undesired side effects, prompting increased interest in developing therapies that specifically eliminate pathogenic bacteria without damaging gut resident flora. To achieve this goal, we developed a strategy utilizing bacterial conjugation to deliver CRISPR-Cas13a programmed to specifically kill S. Typhimurium. This system produced pathogen-specific killing based on CRISPR RNA (crRNAs) targeting endogenous transcripts in pathogens and was shown to be effective in both in vitro and in vivo experiments. Additionally, the system can be readily delivered by conjugation and is adaptable for targeting different pathogens. With further optimization and improvement, the system has the potential to be used for biotherapy and microbial community modification.

Extraction and characterization of bovine collagen Type V and its effects on cell behaviors.

Collagen Type V (Col. V) plays an essential role in cell behaviors and has attracted increasing attention in recent years. High-purity Col. V is needed for evaluating its biological properties. In this research, the enzymatic hydrolysis process was combined with ultrafiltration to purify Col. V from the bovine cornea. The purity of Col. V was determined to be above 90% by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography methods. The effect of Col. V on cell behaviors was evaluated. The circular dichroism spectroscopy results demonstrated that the extracted Col. V exhibited a complete triple helix structure. SDS-PAGE suggested that the molecular weight of Col. V was 440 kDa. The self-assembly experiment revealed that the proportion of Col. V in the collagen mixture can affect the Col. I fiber diameter. The cell culture results implied that Col. V can inhibit fibroblasts (L929) proliferation. The L929 showed maximum mobility when the addition of Col. V was 30%. Thus, Col. V has the effect of inhibiting L929 proliferation and promoting migration. The high-purity Col. V provides useful information for further understanding its biological implications.

Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry.

A method for quantifying the bovine collagen type V (Col. V) was established based on high-performance liquid chromatography coupled to mass spectrometry by the marker peptide external standard. High-purity Col. V was extracted by the acid-enzyme hydrolysis process, and the marker peptide of Col. V was identified by LCQ mass spectrometry as GPAGPMGLTGR. A broad linear range (0.01-5.00 µg/mL) with a correlation coefficient of 0.9984 was achieved, and the limit of detection and limit of quantification were found to be 3.00 × 10-3 and 6.25 × 10-3 µg/mL, respectively. The method precision was 1.49%. The recovery rate was determined as 97.1-109.6% with a relative standard deviation less than 5%. The proposed method was successfully applied for the determination of Col. V contents in the bovine heart, lung, and cornea, which were 0.72 ± 0.01%, 0.23 ± 0.01%, and 2.89 ± 0.00%, respectively. The results show that the proposed method is more suitable for measuring the content of Col. V in tissue samples compared with the enzyme-linked immunosorbent assay. The marker peptide method has high accuracy and great reproducibility, and will lay a foundation for the extraction and application of Col. V. Impact statement The accurate quantitative method for collagen type V (Col. V) is particularly important in scientific research, disease diagnosis and treatment, and industrial production. In this article, we proposed a high-performance liquid chromatography coupled to mass spectrometry method based on the external standard marker peptide to quantify bovine Col. V. This method shows a higher accuracy and recovery rate than enzyme-linked immunosorbent assay (ELISA), indicating that it is more suitable for measuring the content of Col. V in tissue samples than ELISA. The establishment of this method has laid a solid foundation for the extraction and application of Col. V.

Biological evaluations of decellularized extracellular matrix collagen microparticles prepared based on plant enzymes and aqueous two-phase method.

For patients with extensive full-thickness burns who do not have sufficient autologous split-thickness skin for skin grafts, the application of biological skin substitutes may be considered. The aim of this study was to find an optimal new type method for the production of a biovital skin substitute based on acellular dermal matrix (ADM) and preclinical evaluations. In this work, 25 methods of ADM production were assessed. The proposed methods are based on the use of the following enzymes: papain, Carica papaya lipase (CPL), and purification using a polymer/salt aqueous two-phase system. The obtained ADM samples were characterized via scanning electron microscopy (SEM), porosity measurement and water vapor transmission test. Results showed that the collagen bundles of ADM microparticles were intact and orderly. Through differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA) and biocompatibility tests, the results indicated that the proportion of papain and CPL was the same and 5 h processing time are the optimum conditions for ADM preparation and the material showed good biocompatibility. Our results suggested that the potential of developing this kind of decellularization process to manufacture ADM scaffolds for clinical application.

Stability of lutein in O/W emulsion prepared using xanthan and propylene glycol alginate.

Lutein is a hydrophobic carotenoid with diverse bioactivities. For encapsulating the molecule in a novel method, we prepared two emulsions from xanthan and propylene glycol alginate at the ratios of 3:7 and 4:6. The instability index and particle size of the emulsions were determined using a stability analyzer and laser particle size analyzer. The influence of crystallization on the emulsions was observed under a polarizing microscope. The effects of centrifugal force and storage on the lutein emulsions were analyzed by measuring the changes in absorbance. The results showed that the emulsion fabricated by xanthan and propylene glycol alginate at the ratio of 4:6 was highly stable, and crystals were dispersed when xanthan and propylene glycol alginate existed. These results revealed that the hydrophobicity and absorption kinetics of emulsifiers would determine the stability of emulsion when the viscosity of emulsifiers reached a certain value, and the stability of emulsions would affect the stability of lutein in the emulsions.

Impact of Surfactants on Nanoemulsions based on Fractionated Coconut Oil: Emulsification Stability and in vitro Digestion.

Functional oils have broad application prospects in functional foods and beverages because of their rich beneficial ingredients and healthier intake. The small droplets of the nanoemulsion enhance the effective delivery, solubility and bioavailability of the various hydrophobic food components. This study used a mixed oil phase of green tea seed oil and fractionated coconut oil, compared the emulsifying properties of natural surfactants: Whey protein isolate, soy lecithin, tea saponin and synthetic surfactant: Tween 80 in the preparation of nanoemulsions by ultrasonic method. In particular, the impact of emulsifier type and concentration, pH, ionic strength, and heat treatment on the mean particle size and ξ-potential were investigated. The long-term storage stability of the fabricated nanoemulsions was also monitored during storage at different temperatures. In addition, the effects of emulsifier type on the bioavailability of nanoemulsions were evaluated. For all nanoemulsions studied, the mean particle size decreasing with increasing emulsifier concentration. Tea saponin and soy lecithin can produce smaller droplets of nanoemulsion than Whey protein isolate. Tea saponin has the same emulsifying ability as Tween 80. Presumably tea saponin-stabilized droplets may be maintained by electrostatic repulsion and steric repulsion. All of the nanoemulsions significantly improved the bioavailability of the mixed oil phase compared to the unemulsified oil phase. This study highlights the potential of natural surfactants in the ultrasonic preparation of nanoemulsions containing functional oils, and provides a basis for the application of natural surfactants and new functional oils in food industry.

Development of Low-calorie Organogel fromsn-2 Position-modified Coconut Oil Rich in Polyunsaturated Fatty Acids.

Coconut oil rich in medium-chain saturated fatty acids was enzymatically modified at the sn-2 position with polyunsaturated fatty acids from fish oil by trans-esterification reaction. The modified coconut oil was combined with gelators (γ-oryzanol and ß-sitosterol) to prepare organogels. The effects of different modified coconut oil contents and γ-oryzanol:ß-sitosterol ratios on thermodynamic and rheological properties, and microstructures of organogels, as well as their relationships, were studied. The results showed that the addition of gelators increased the hardness, solid fat content, and oil binding capacity of organogels. In addition to its highest melting point and enthalpy change, the organogel containing γ-oryzanol:ß-sitosterol (6:4) had the best texture properties that closely resemble the crystalline structure properties. Moreover, the developed organogels had the properties of a pseudoplastic fluid, as described by the power law equation. G' of organogel was found to be significantly higher than G'', which indicates that the organogel remains in solid state. The analysis of crystal morphology showed that the crystal of organogels were clusters, consisting of dense three-dimensional network of gel.

Fatty Acid Profiles of Triacylglycerols and Phospholipids of Sea-Cage Cultured Trachinotus blochii: A Comparative Study of Head, Viscera, Skin, Bone, and Muscle.

Trachinotus blochii (golden pompano) is an economically important cultured marine fish that is rich in polyunsaturated fat. The fatty acid profiles of triacylglycerols and phospholipids of T. blochii tissues were analyzed. Lipid contents in tissues followed the order of viscera (28.27%) > skin (17.25%) ≈ bone (16.18%) ≈ head (15.12%) > muscle (7.38%). In triacylglycerols (TAG), saturated fatty acids (palmitic acid predominated) were preferentially esterified in the sn-2 position, and monounsaturated fatty acids (oleic acid predominated) were mainly distributed in the sn-1, 3 positions. The numbers of n-3 polyunsaturated fatty acids (PUFA) in the sn-2 position were significantly greater than those in positions 1 and 3, while the n-6 PUFA were mainly found in the sn-1, 3 positions. Muscle TAG had a higher content of EPA+DHA (3.90%) than did the other tissues. In phosphatidylethanolamines, EPA+DHA in muscle (6.76%) predominated over that in other tissues. In phosphatidylcholines, EPA+DHA in bone (6.50%) predominated. This study is a guide for the biochemical and nutritional values of the T. blochii species and can be useful for further investigation of industrial applications. PRACTICAL APPLICATION: The high proportion of sn-2-palmitic acid (greater than 50%) is close to that of breast milk and the n-3 PUFA, especially EPA and DHA have the preferential distribution in the sn-2 position of triacylglycerol. Hence, golden pompano is an excellent dietary component for human nutrition and health.

Optimization of Microencapsulation of Human Milk Fat Substitute by Response Surface Methodology.

Human milk fat substitutes (HMFS) are rich in polyunsaturated fatty acids which upon microencapsulation, can be used as a source of high quality lipids in infant formula. The response surface methodology (RSM) was employed to optimize the microencapsulation condition of HMFS as a functional product. The microencapsulation efficiency (MEE) of microencapsulated HMFS was investigated with respect to four variables including concentration of soy lecithin (A), ratio of demineralized whey powder to malt dextrin (B), HFMS concentration (C), and homogenizing pressure (D). The optimum conditions for efficient microencapsulation of HMFS by the spray drying technique were determined as follows: the amount of soybean lecithin-0.96%, ratio of desalted whey powder to malt dextrin-2.04:1, oil content-17.37% and homogeneous pressure-0.46MPa. Under these conditions, the MEE was 84.72%, and the basic indices of the microcapsules were good. The structure of the microcapsules, as observed by scanning electron microscopy (SEM), revealed spherical, smooth-surfaced capsules with diameters ranging between 10-50 µm. Compared with HFMS, the peroxide value (POV) and acid value (AV) of the microcapsule were significantly lower during storage indicating that the microencapsulation process increases stability and shelf life. Infrared spectroscopic analyses indicated that HFMS had the same characteristic functional groups as the oil extracted from microcapsules. Simulated in vitro digestion revealed that the microcapsules were digested completely within 2h with maximum lipid absorption rate of 64%. Furthermore, these results advocate the embedding process of HFMS by RSM due to its efficacy.

Effects of Ultrasonic Parameters on the Crystallization Behavior of Virgin Coconut Oil.

Crystallization behavior of virgin coconut oil (VCO) in the absence and presence of ultrasonic treatment under a temperature gradient field was investigated. The effects of ultrasonic parameters on the crystallization behavior of VCO were studied by differential scanning calorimetry, ultraviolet/visible spectrophotometry and polarized light microscopy. The thermal effect of the ultrasonic treatment was also increased at higher power levels. Therefore, the optimal power level was determined at approximately 36 W. Induction time reduced evidently and the crystallization rate was accelerated under ultrasonic treatment at crystallization temperature (Tc) above 15°C. However, no significant difference in induction time was noted at 13°C. The result of morphological studies showed that the growth mechanism of crystals was significantly changed. Meanwhile, smaller and uniform crystals were produced by the ultrasonic treatment. This study shows a novel technique to accelerate the crystallization rate and alter the growth mechanism of VCO crystals.

[Immunity and EGFPC3 expression of recombinant attenuated salmonella typhimurium carrying plasmid pcDNA3s and EGFPC3 in mice].

AIM: To study the immune characterization of HBsAg DNA recombinant attenuated salmonella typhimurium vaccine and the expression of enhanced green fluorescence protein gene pEGFPC3 in mice. METHODS: Plasmid pcDNA3s and pEGFPC3 was transformed into attenuated salmonella typhimurium SL8786 by electricity instrument to construct recombinant vaccine SL8786/EGFPC3 and SL8786/pcDNA3s. Mice were immunized with the recombinant vaccine and the quantity of anti-HBs antibody in the sera was measured by time resolved fluorescence immunoassay(TRFIA), While the cytotoxocity of CTL was measured by LDH release assay. RESULTS: It produced strong specificity CTL response after taking SL8786/pcDNA3s orally to mice and anti-HBs was highest in 11 weeks. EGFPC3 expression in two mice spleen cells was detected by flow cytometry(FCM). EGFP masculine ratio is 19.20% and 17.36% respectively. but SL8786/pcDNA3 in two mice spleens cell EGFP masculine ratio only is 1.95% and 1.63%. The mice was taken SL8786/EGFPC3 orally after three weeks, fluorescence expression in its liver, spleen, kidney, chest gland, duodenum, muscle, etc. were observed. CONCLUSION: HBV DNA vaccine can induce specific cellular and humoral immune responses in vivo in mice by oral. Recombinant attenuated salmonella typhimurium SL8786/EGFPC3 we constructed. It has green fluorescence expression in mice's partial organization, and it offers a model for research gene engineering vaccine.

Antioxidant and protective effect of an oleanolic acid-enriched extract of A. deliciosa root on carbon tetrachloride induced rat liver injury.

The ethanol-water extract of A. deliciosa root (EEAD) was fractionated into n-hexane (EEAD-He), ethyl acetate (EEAD-Ea), n-butanol (EEAD-Bu) and aqueous (EEAD-Aq) fractions according to their different polarity and solubility. Among the four extracts, it was found that EEAD-Bu was enriched with oleanolic acid (OLA). The antioxidant and hepatoprotective activities of various EEAD fractions and OLA were carefully investigated by the methods of ferric thiocyanate (FTC) and thiobarbituric acid (TBA), as well as the model of CCL4-induced liver toxicity in rats. The results showed that the EEAD-Bu had higher in vitro antioxidant and in vivo hepatoprotective activities than those of the other types of extracts (p< 0.05). When the CCL4-induced rats were treatment with 120 mg/kg EEAD-Bu, the activities of alanine transaminase (ALT) and aspartate transanimase (AST) in rat serum decreased 90 % and 81 %, respectively, as compared with those of the CCL4 control rats. Furthermore, the lipid peroxidation (MDA) decreased 42 % and glutathione (GSH) increased 114 % in the rats liver homogenate, as compared with those of the control. The results also indicated that the hepatoprotective activity of the EEAD-Bu (at the dose of 120 mg/kg) was higher than that of the reference drug silymarin (at the dose of 60 mg/kg), and OLA acted as an important role in dose-dependent protection against CCL4 hepatotoxicity. The findings indicate that the OLA-enriched EEAD-Bu extract had significant and concentration dependent hepatoprotective effect for the carbon tetrachloride induced rat liver injury.