Casein Hydrolysate Research Articles

Microbiota-dependent proteolysis of gluten subverts diet-mediated protection against type 1 diabetes

Diet and commensals can affect the development of autoimmune diseases like type 1 diabetes (T1D). However, whether dietary interventions are microbe-mediated was unclear. We found that a diet based on hydrolyzed casein (HC) as a protein source protects non-obese diabetic (NOD) mice in conventional and germ-free (GF) conditions via improvement in the physiology of insulin-producing cells to reduce autoimmune activation. The addition of gluten (a cereal protein complex associated with celiac disease) facilitates autoimmunity dependent on microbial proteolysis of gluten: T1D develops in GF animals monocolonized with Enterococcus faecalis harboring secreted gluten-digesting proteases but not in mice colonized with protease deficient bacteria. Gluten digestion by E.faecalis generates Tcell-activating peptides and promotes innate immunity by enhancing macrophage reactivity to lipopolysaccharide (LPS). Gnotobiotic NOD Toll4-negative mice monocolonized with E.faecalis on an HC+ gluten diet are resistant to T1D. These findings provide insights into strategies to develop dietary interventions to help protect humans against autoimmunity.

Enhancing the Production of Hydrolytic Enzymes in Elicited Tamarillo (Solanum betaceum Cav.) Cell Suspension Cultures.

Plant cell suspension cultures are widely used as a tool for analyzing cellular and molecular processes, metabolite synthesis, and differentiation, bypassing the structural complexity of plants. Within the range of approaches used to increase the production of metabolites by plant cells, one of the most recurrent is applying elicitors capable of stimulating metabolic pathways related to defense mechanisms. Previous proteomics analysis of tamarillo cell lines and cell suspension cultures have been used to further characterize and optimize the growth and stress-related metabolite production under in vitro controlled conditions. The main objective of this work was to develop a novel plant-based bioreactor system to produce hydrolytic enzymes using an elicitation approach. Based on effective protocols for tamarillo micropropagation and plant cell suspension culture establishment from induced callus lines, cell growth has been optimized, and enzymatic activity profiles under in vitro controlled conditions characterized. By testing different sucrose concentrations and the effects of two types of biotic elicitors, it was found that 3% (w/v) sucrose concentration in the liquid medium enhanced the production of hydrolytic enzymes. Moreover, casein hydrolysate at 0.5 and 1.5 g/L promoted protein production, whereas yeast extract (0.5 g/L) enhanced glycosidase activity. Meanwhile, chitosan (0.05 and 0.1 g/L) enhanced glycosidases, alkaline phosphates, and protease activities.

Enzymatic preparation of casein hydrolysates with high digestibility and low bitterness studied by peptidomics and random forests analysis.

Enzymatic hydrolysis can not only increase the digestibility of casein, but also cause bitterness. This study aimed to investigate the effect of hydrolysis on the digestibility and bitterness of casein hydrolysates and provided a novel strategy for the preparation of high-digestibility and low-bitterness casein hydrolysates based on the release pattern of bitter peptides. Results showed that with the increase of the degree of hydrolysis (DH), the digestibility and bitterness of hydrolysates increased. However, the bitterness of casein trypsin hydrolysates rapidly increased in the low DH range (3%-8%), while the bitterness of casein alcalase hydrolysates rapidly increased in a higher DH range (10.5%-13%), indicating the discrepancy in the release pattern of bitter peptides. Peptidomics and random forests revealed that peptides containing >6 residues with hydrophobic amino acids (HAAs) at the N-terminal and basic amino acids (BAAs) at the C-terminal (HAA-BAA type) obtained from trypsin contributed more to the bitterness of casein hydrolysates than those containing 2-6 residues. On the other hand, peptides containing 2-6 residues with HAAs at both N- and C-terminals (HAA-HAA type) released by alcalase contributed more to the bitterness of casein hydrolysates than those containing >6 residues. Furthermore, a casein hydrolysate with a significantly lower bitter value containing short-chain HAA-BAA type peptides and long-chain HAA-HAA type peptides from the combination of trypsin and alcalase was obtained. The digestibility of the resultant hydrolysate was 79.19% (52.09% higher than casein). This work is of great significance for the preparation of high-digestibility and low-bitterness casein hydrolysates.

Four novel sleep-promoting peptides screened and identified from bovine casein hydrolysates using a patch-clamp model in vitro and Caenorhabditis elegans in vivo.

Bovine casein hydrolysates (CHs) have demonstrated sleep-promoting activities. However, only few peptides were identified from CHs with sleep-promoting effects. In this work, an in vitro model based on the electrophysiology of brain neurons was established for the evaluation of sleep-promoting effects. Based on this model, four novel peptides were systematically separated from CH. Compared with the control group, the action potential (AP) inhibitory rate of four peptides increased by 38.63%, 340.93%, 233.28%, and 900%, respectively, and the membrane potential (MP) change rate of four peptides increased by 319.78%, 503.09%, 381.22%, and 547.10%, respectively. These results suggested that four peptides have sleep-promoting activities. Furthermore, Caenorhabditis elegans (C. elegans) sleep behavior results indicated that all the four peptides could significantly increase the total sleep duration, the motionless sleep duration of C. elegans, implying that these four peptides can significantly improve sleep. The LC-MS/MS results showed that the primary structures of these novel peptides were HQGLPQEVLNENLLR (αs1-CN, f8-22), YKVPQLEIVPNSAEER (αs1-CN, f104-119), HPIKHQGLPQEVLNENLLR (αs1-CN, f4-22), and VPQLEIVPNSAEER (αs1-CN, f106-119). Overall, this study revealed that the four novel sleep-promoting peptides identified were strong candidates as potential functional ingredients in the development of sleep-promoting products.

Enhancement of callogenesis from plumular explants of coconut (Cocos nucifera) via exogenous supplementation of amino acids and casein hydrolysate

The role of exogenous supplementation of amino acids and casein hydrolysate in enhancing callogenesis from plumular explants of coconut (

Establishing a high-efficiency <i>in vitro</i> regeneration system for <i>Agrobacterium</i>-mediated transformation in <i>Fragaria nilgerrensis</i>

<italic>Fragaria nilgerrensis</italic> Schlechtendal ex J. Gay, a diploid wild strawberry, has many excellent characteristics such as fruit with white color and peach fragrance, and strong disease resistance. However, the lack of an efficient and stable regeneration and genetic transformation system for <italic>F. nilgerrensis</italic> has largely limited the functional studies of related genes governing excellent traits. In this study, a regeneration system for <italic>F. nilgerrensis</italic> leaf discs was developed by optimizing factors such as different hormone combinations, dark culture times and casein hydrolysate (CH) concentrations, with an average regeneration rate of 97.3% at 45 d of culture. By paraffin section observation of callus with different colors induced from the leaf disc regeneration, light yellow callus was determined to be embryogenic and the regenerative pathway was identified as indirect organogenesis. Based on this, an average transformation percentage of 8.67% was achieved by screening kanamycin concentration and referring to transformation procedures described by predecessors. PCR-positive transformants were obtained within 4-5 months by confirmation of PCR and histochemical GUS, and transgene integration was identified by transformants regeneration. The establishment of efficient regeneration system provided a feasible platform for genetic transformation, which laid the foundation for further gene functional studies in <italic>F. nilgerrensis</italic>.

Isolation, Characterization, and Evaluation of Protease Activity of Crude Bromelain of Pineapple Peel, Core, and Crown from Subang District, Indonesia.

Bromelain is distributed to all parts of pineapple (Ananas comusus (L.) Merr), including the peel, core, and crown, which are agricultural waste that has not been utilized properly. The purpose of this study was to determine the character and protease activity of crude bromelain from the Indonesian pineapple peel, core, and crown. The pineapple was collected from Subang district, West Java Province, Indonesia. The three crude bromelains were obtained through the precipitation process with ethanol, then a protein qualitative and quantitative analysis was performed. Protease activity was determined by measuring the tyrosine produced from casein hydrolysis. The characters of crude bromelains were determined by evaluating the protease activity in various pH, temperature, and substrate concentration. One-way analysis of variance was conducted to analyze the data statistically. The three crude bromelains can be isolated from the peel, core, and crown of pineapple fruit with protease activity in the range of 38.32-46.78 units. Crude bromelains have an optimum temperature of 55°C for the peel and core and 35°C for the crown. All crude bromelains have an optimum pH of pH 7. The three crude bromelains have Vmax in the range of 140.85 to 285.71 units and KM in the range of 15298.59 to 18370.86 ppm. It concluded that the three crude bromelains have protease activity with the specific character and kinetic parameter.

Casein-phenol interactions occur during digestion and affect bioactive peptide and phenol bioaccessibility.

Casein (CN) represents many proline residues that may bind polyphenols. Some evidence exists of CN-polyphenols interaction in model systems. The formation of such interactions upon digestion and the effects on CN digestibility and potential functionality due to the release of bioactive peptides are obscure. This study aimed to explore the interactions of CN with different phenol compounds under digestive conditions and monitor how they affect the bioaccessibility of phenol compounds and bioactive peptides. CN or CN hydrolysate and phenol compounds such as chlorogenic acid, ellagic acid, catechin, green tea extract, and tea extract, singularly or in combination with CN were digested in vitro. Total antioxidant capacity (TAC), degree of hydrolysis, and bioactive peptide formation were assessed in the samples collected through the digestion. The results showed that bioaccessible TAC was 1.17 to 1.93-fold higher in CN co-digested with phenol compounds than initially due to a higher release of antioxidant peptides in the presence of phenolic compounds. However, TAC values in the intestinal insoluble part of CN-phenol digests were higher than the initial, indicating that such interactions may be functional to transport phenols to the colon. Bioactive peptide release was affected by the phenol type (catechins were the most effective) as well as phenol concentration. As an opioid peptide released from β-CN, β-casomorphin formation was significantly influenced by the co-digestion of CN with phenol compounds. This study confirmed the possible CN-phenol interaction during digestion, affecting bioactive peptide release.

Structure relationship of non-covalent interactions between lotus seedpod oligomeric procyanidins and glycated casein hydrolysate during digestion.

Procyanidin-amino acid interactions during transmembrane transport cause changes in the structural and physical properties of peptides, which limits further absorption of oligopeptide-advanced glycation end products (AGEs). In this study, glycated casein hydrolysates (GCSHs) were employed to investigate the structure and interaction mechanism of GCSH with lotus seedpod oligomeric procyanidin (LSOPC) complexes in an intestinal environment. LSOPC can interact with GCSH under certain conditions to form hydrogen bonds and hydrophobic interactions to form GCSH-LSOPC complexes. Results showed that procyanidin further leads to the transformation of a GCSH secondary structure and the increase of surface hydrophobicity (H0). The strongest non-covalent interaction between GCSH and (-)-epigallocatechin gallate (EGCG) was due to the polyhydroxy structure of EGCG. Binding site analysis showed that EGCG binds to the internal cavity of P1 to maintain the relative stability of the binding conformation. The antioxidant capacity of GCSH was remarkably elevated by GCSH-LSOPC. This study will provide a new reference for the accurate control of oligopeptide-AGEs absorption by LSOPC in vivo.

Optimization of Protease Production Using Agro Wastes by Aspergillus spp. In A Solid-State Fermentation

gricultural wastes are highly nutritious and facilitate microbial growth and metabolism. Most agro-wastes are lignocellulosic in nature, composed of lignin, hemicellulose and cellulose. Agricultural residues can be used for the production of various value-added products, such as industrially important enzymes. Extracellular proteases have high commercial value and multiple applications in detergents, food, dairy, pharmaceutical, leather, diagnostics, waste management and several recovery industries. Aspergillus oryzae was isolated from rice farmland, Rigasa, Kaduna. It was screened for protease production using skimmed milk agar assay with a wider clear zone of casein hydrolysis (halo zone). The main objectives of this study were to isolate, characterized, and optimize the production conditions by Aspergillus oryzae under solid state fermentation (SSF) using locally available agricultural wastes used as substrate. The results showed that the optimum conditions for maximum protease production are 84 hours of incubation; pH 6.0; temperature of 35ᵒC; 3% inoculum size; peptone as Nitrogen source and rice husk as the best fermentation substrate. The highest protease activity of rice husk was 1350 Ug−1 of dry solids compared with an activity of corn cob 1005 Ug−1 respectively. Protease was produced from Aspergillus oryzae with broad substrate specificity, high pH and temperature stability.

Effect of plasmin on casein hydrolysis and textural properties of rennet-induced model cheeses

Plasmin plays an important role in casein hydrolysis during cheese ripening, which may influence the properties of the casein network and the texture of the final product. In view of this, the relation between plasmin-induced casein hydrolysis and textural changes of cheese during ripening was investigated in this study. Four batches of model cheese with different concentrations of added plasmin (0, 0.4, 0.6 and 1.0 μL/g milk) were prepared, and were stored for 12 weeks at 16 °C. During this period, plasmin activity, casein hydrolysis, textural properties and other compositional characteristics (pH, dry matter) were determined. Our results show that the addition of plasmin had significant effect on both the degree and the pattern of proteolysis. As a result, cheeses with different plasmin content showed different textural properties. With increased plasmin concentration, Young’s modulus, hardness, resilience and cohesion decreased, while brittleness increased. All textural properties showed linear relations with the degree of casein hydrolysis, and logarithmic relations with the percentage of intact casein fractions. At the beginning of ripening, only slight changes in textural properties were found, although a substantial part (40–60 %) of the casein fractions was already been broken down. When ripening progressed, ongoing proteolysis significantly weakened the protein network and consequently led to noticeable textural changes. Model cheeses became softer, more brittle and less elastic. The knowledge gained from this study provide new insights in the changes of different textural parameters of model cheese. This will help to optimize the existing products and create new ones.

THE EFFECT OF INDOLYLBUTYRIC ACID ON THE REGENERATION OF MAIZE HYBRIDS IN VITRO

Еру Optimization of the regeneration potential in vitro for maize (Zea mays L.) hybrids with the participation of perspective inbreds is an actual task for biotechnological maintenance of this important crop breeding process.&#x0D; The purpose of the research was the investigation of the effect of the auxin phytohormone — indolylbutyric acid (IBA) on the in vitro regeneration capacity of maize hybrids. The task of the work was to determine the effect of IBA in concentrations of 0.1 and 0.2 mg / L on the total regeneration frequency from type II callus tissue, as well as on the number of regenerated plants per 100 calli. The material of the study was 90-day type II callus tissue of maize hybrids between inbreds DK633, DK3070, DK236 and DK267 of Lancaster germplasm from the Dnipro breeding program and inbreds with high regeneration capacity A188, Chi31 and PLS61. The method of cell, tissue and organ culture in vitro was used. The initiation of plant regeneration from callus tissue was performed on the mineral MS medium, supplemented with 100 mg / L inositol, 100 mg / L casein hydrolysate, 690 mg / L L-proline, 20 g / L sucrose, 6 g / L agar, and 0.1 or 0.2 mg / L IBA.&#x0D; It was found that IBA significantly affected the maize hybrids’ regeneration capacity. With 0.1 mg / L IBA in the medium, the total regeneration frequency increased up to 32.1 ± 7.7 %, compared to the control without IBA (15.6 ± 5.3 %) and to 0.2 mg / L IBA (17.0 ± 5.5 %). IBA (0.1 mg / L) contributed also to the increase of the number of regenerated plants up to 19.5 ± 4.3 pcs/100 calli, compared with their number without IBA (9.7 ± 4.3 pcs/100 calli) and with 0.2 mg / L IBA (6.5 ± 3.6 pcs/100 calli). The genotype of callus tissue significantly affected the regeneration ability of the hybrids. The highest number of regenerated plants per 100 calli under 0.1 mg / L IBA was obtained in hybrid DK236xChi31 (68.8 pcs/100 calli), the smallest one — in PLS61xDK236 (4.3 pcs/100 calli). The highest total regeneration frequency was observed for DK236xChi31 (75.0 %), and the lowest —for PLS61xDK236 (4.4 %) as well.&#x0D; In the perspective the obtained outcomes can be used to optimize the biotechnology of maize callusogenesis and plant regeneration in vitro and to increase somaclonal production from long-term cultivated calli for application in breeding process.

Large-Scale In Vitro Multiplication and Phytochemical Analysis of Himantoglossum affine (Boiss.) Schltr.: An Endangered Euro-Mediterranean Terrestrial Orchid

Himantoglossum affine is a threatened terrestrial orchid. We aimed to optimize asymbiotic seed germination and direct embryogenesis and to analyze the phytochemical profile and physico-biochemical analysis of leaf and tuber. The individual use of organic nitrogen compounds resulted in higher germination efficiencies, while the shortest times to germination were observed using coconut water plus casein hydrolysate. Plantlets grown on media supplemented with pineapple juice and peptone had the highest plantlet length and weight. For embryogenesis, the highest regeneration rate (44%) and embryo number/explant (10.12 ± 2.08) were observed in young protocorm-like body (PLB) explants with 0.5 mg/L naphthalene acetic acid (NAA) and 1 mg/L thidiazuron (TDZ). During the acclimatization process, the scattered vascular tubes converted to fully developed vascular tissues, ensuring maximum sap flux. Gas chromatography–mass spectrometry analysis identified 1,2,3-propanetriol, monoacetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and 2-butenedioic acid, 2-methyl-, (E)- as the most prevalent compounds. We reported higher contents of total phenolics and flavonoids and antioxidant activity compared to other terrestrial orchids. The glucomannan content (36.96%) was also higher than starch content (31.31%), comparable to those reported in other tuberous orchids. Based on the fragmentation of H. affine populations in the Middle East and Euro-Mediterranean countries due to over-harvesting, climate change, and/or human impact, our procedure offers a tool for the re-introduction of in vitro-raised plants to threatened areas.

Induction of callus and establishment of cell suspension and effects on tea polyphenols in the cell suspension system of tea plants by PGRs

The effects of different plant growth regulators (PGRs) on the accumulation of tea polyphenols were investigated using the sterile leaves of the ‘Yunkang-10’ tea seedlings in test tubes as explants. Using the MS media with a sucrose concentration of 30 g/L as the basal medium, the leaf callus was induced and subcultured in vitro. The medium containing MS + 1.0 mg/L 2,4-D + 0.8 mg/L 6-BA was the most efficient for callus induction from leaf explants, with an induction rate of 78.89%. The loose calli were subcultured in the medium containing MS + 0.5 mg/L 2,4-D + 0.3 mg/L KT. The loose calli were transferred into liquid MS medium containing 2.0 mg/L 2,4-D, 0.3 mg/L KT, and casein hydrolysate (CH) 1000 mg/L. A stable cell suspension was obtained in the ninth generation with an initial inoculum of 80 g/L. The suspension was cultured in the dark at 25 ± 2°C with 110 rpm, with the medium refreshed every 16 days. Further, the amount of tea polyphenols in the tea suspension reached the maximum in the presence of 5.0 mg/L MeJA, 1.0 mg/L SA, 1.0 mg/L GA3, 1.0 mg/L ETH and 200 μmol/L ABA, with the growth rate of 154.68% on day 4, 172.81% on day 6, 213.55% on day 2, 104.48% on day 12 and 220.77% on day 2. The ABA induced the maximum accumulation of tea polyphenols in the shortest time. In this study, a stable cell suspension system was established from the callus of tea leaves. The findings suggest that the biosynthesis of tea polyphenols might be mediated mainly via the ABA signaling pathway, with a possible crosstalk between MeJA, SA, GA3, ETH and ABA. In addition, the extraction time and amount of tea polyphenols in vitro was controlled by adding different PGRs.

Casein Hydrolysate and Casein–Iron Chelate as Natural Bioactive Compounds for Yoghurt Fortification

The aim of the study was to apply the bioactive components casein hydrolysate and casein–iron chelate to yoghurt and to analyze their influence on product parameters. Casein was hydrolyzed with the non-commercial protease obtained from Yarrowia lipolytica yeast and subjected to chelating process with Fe2+ ions. Both preparations were introduced to yoghurt production as additives for elevation of dry matter by 2%. The control yoghurt was produced with the addition of skim milk powder. In the prepared experimental yoghurts, the content of dry matter [DM], protein, fat, as well as iron contents were determined. The conducted studies showed that in all analyzed yogurts, DM content was at a similar level, while the protein content was significantly (p &lt; 0.05) higher in the variant containing 2% casein–iron chelate. In yoghurts with the casein–iron chelate as a bioactive compound, the iron concentration was about 61.5–123 times higher than in yoghurt with the addition of skim milk powder. It was shown that during storage the addition of casein–iron chelate positively influenced the number of analyzed microbe groups.

Optimization of Bacillus subtilis NRC1 growth conditions using response surface methodology for sustainable biosynthesis of gold nanoparticles

Gold nanoparticles (AuNPs) have different unique properties and a wide range of applications in different fields. Thereby, there is a growing urgency for the production of AuNPs using a safe and an economic method. In this study, optimization of fermentation conditions by Bacillus subtilis NRC1 for extracellular AuNPs synthesis using response surface methodology was achieved. The data obtained from Plackett–Burman design followed by Box–Behnken design indicated the accuracy and reliability of the model and it could be used to navigate the design space with a reasonable accuracy. Numerical optimization of Bacillus subtilis NRC1 active extracellular filtrate production, showed the optimum conditions of 0.74% (w/v) casein hydrolysate, 3.99% (w/v) dextrin, 47 × 106 CFU/ml inoculum size at pH 7.76 and 25 ^circC to give the maximum AuNPs biosynthesis. The model was highly valid and the obtained data had a confidence factor of 98.48%. Statistical optimization resulted in a 2.6-fold increase in AuNPs production compared with that of the non-optimized medium.

In vitro asymbiotic seed germination, protocorm formation, and plantlet development of Orchis simia Lam.: A threatened terrestrial orchid species

• Seed germination and plantlet growth were impacted by organic constituents and nitrogen sources. • Individual application of organic nitrogen compounds led to higher germination efficiencies. • The present protocol could be used for large-scale in vitro micropropagation of terrestrial orchids. The present study examined the seed morphometric and viability, asymbiotic seed germination, protocorm formation, and plantlet growth in Orchis simia Lam., an at-risk Euro-Mediterranean terrestrial orchid species. Furthermore, tissue histology pattern in vitro grown plantlets and field-established was compared. After one month, shoot and root primordia started to grow on developing protocorms and small plantlets became visible after 2–3 subculturing. Asymbiotic in vitro seed germination and plantlet growth attributes were significantly ( P <0.05) impacted by both type of main organic constituent and nitrogen sources. The seeds sown on media supplemented with pineapple juice (PJ) and casein hydrolysate (CH) had the highest germination percentage. In terms of required days to see the first signs of germination, CW+AV treatment brings about the fastest germination (6.8 ± 0.20 days). Protocorms grown on the media comprising PJ in combination with Aminoven (AV) or casein hydrolysate (CH) or Pep showed higher dimensions and therefore higher weight (% 450 increase) compared to protocorms grown on other media. Altogether, treatments with higher germination percentage, faster germination, and bigger protocorms finally resulted in better plantlet growth. Acclimatization of in vitro grown plantlets occurred by converting scattered vascular tubes into fully developed xylem and phloem vessels facilitating sap flow. Taking into account the fragmentation of O. simia populations in the Middle East and the Euro-Mediterranean basin mainly due to tuber overharvesting and climate change, the present procedure could be used for large-scale multiplication of in vitro raised seedlings to re-introduce into endangered regions.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation

During the thermal processing of dairy products, the Maillard reaction occurs between milk proteins and lactose, resulting in the formation of various products including glycated proteins. In this study, lactose-glycated casein was generated through the Maillard reaction between casein and lactose and then hydrolyzed by a trypsin preparation. The anti-inflammatory effect of the resultant glycated casein hydrolysate (GCH) was investigated using the lipopolysaccharide (LPS)-sitmulated rat intestinal epithelial (IEC-6) cells as a cell model and corresponding casein hydrolysate (CH) as a control. The results indicated that the preformed glycation enabled lactose conjugation to casein, which endowed GCH with a lactose content of 12.61 g/kg protein together with a lower activity than CH to enhance the viability value of the IEC-6 cells. The cells with LPS stimulation showed significant inflammatory responses, while a pre-treatment of the cells with GCH before LPS stimulation consistently led to a decreased secretion of three pro-inflammatory mediators, namely, IL-6, IL-1β and tumor necrosis factor-α (TNF-α) but an increased secretion of two anti-inflammatory mediators, including IL-10 and transforming growth factor-β (TGF-β), demonstrating the anti-inflammatory potential of GCH in LPS-stimulated cells. In addition, GCH up-regulated the expression of TLR4, p-p38, and p-p65 proteins in the stimulated cells, resulting in the suppression of NF-κB and MAPK signaling pathways. Collectively, GCH was mostly less efficient than CH to exert these assessed anti-inflammatory activities in the cells and more importantly, GCH also showed an ability to cause cell inflammation by promoting IL-6 secretion and up-regulating the expression of TLR4 and p-p65. The casein lactose-glycation of the Maillard-type was thereby concluded to attenuate the anti-inflammatory potential of the resultant casein hydrolysate. It is highlighted that the casein lactose-glycation of the Maillard-type might cause a negative impact on the bioactivity of casein in the intestine, because the glycated casein after digestion could release GCH with reduced anti-inflammatory activity.

Fulvivirga sedimenti sp.nov, isolated from the sediment of oceanic tidal zone.

A yellow-orange, rod-shaped (0.3-0.4 × 2.2-3.8µm), Gram-stain-negative, non-motile, atrichous bacterium, designated strain 1062T, was isolated from tidal zone sediment collected from Xiaoshi Island, Weihai, Shandong Province, China. Growth of strain 1062T occurred at 15-43°C (optimum, 37°C), pH 6.0-9.5 (optimum, pH 7.5) and with 0.5-7.0% (w/v) NaCl (optimum, 2.0-3.0%). Nitrate was not reduced to nitrite or nitrogen. Positive for catalase and oxidase activity, as well as the hydrolysis of casein, DNA, starch and Tweens 20, 40, 60, but negative for hydrolysis of alginate, CM-cellulose, agar and Tweens 80. The 16S rRNA gene sequence comparisons indicated that strain 1062T had a sequence similarity of 96.2% with Fulvivirga lutimaris KCTC 42720T and 94.3% with Fulvivirga kasyanovii KCTC 12832T. Phylogenetic analysis showed that strain 1062T belonged to the genus Fulvivirga. The genomic DNA G + C content was 45.1mol%. The ANI values of strain 1062T in comparison to F. kasyanovii KCTC 12832T and F. lutimaris KCTC 42720T were all lower than 70%. The dDDH values of strain 1062T in comparison to F. kasyanovii KCTC 12832T and F. lutimaris KCTC 42720T were all lower than 20%. The average amino acid identity values of strain 1062T in comparison to F. kasyanovii KCTC 12832T and F. lutimaris KCTC 42720T were all lower than 60%. The major fatty acids (> 5%) of strain 1062T were iso-C15:0, iso-C15:0 G, iso-C15:0 3OH, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c/C16:1ω6c). The polar lipids consist of an aminolipid, phosphatidylethanolamine, phosphatidyglycerol and diphosphatidylglycerol. The major respiratory quinone was MK-7. Based on the results of physiological, biochemical and chemotaxonomic characteristics, the strain 1062T (= KCTC 72868T = MCCC 1H00499T) was identified as a new species of the genus Fulvivirga and the name Fulvivirga sedimenti sp. nov, is proposed.