Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

Small Molecule Oligopeptides Isolated from Walnut (Juglans regia L.) and Their Anti-Fatigue Effects in Mice.

Walnut (Juglans regia L.) is unique for its extensive biological activities and pharmaceutical properties. There are few studies on walnut oligopeptides (WOPs), which are small molecule peptides extracted from walnuts. This study aimed to evaluate the anti-fatigue effects of WOPs on ICR mice and explore the possible underlying mechanism. Mice were randomly divided into four experimental sets and each set of mice were then randomly divided into four groups. The vehicle group was administered distilled water, and the three WOP intervention groups were orally administered WOP solution at a dose of 110, 220, and 440 mg/kg of body weight, respectively. After 30 days of WOP intervention, the anti-fatigue activity of WOPs were evaluated using the weight-loaded swimming test and by measuring the change of biochemical parameters, glycogen storage and energy metabolism enzymes, anti-oxidative capacity and mitochondrial function. It was observed that WOPs could significantly prolong the swimming time, decrease the accumulation of lactate dehydrogenase (LDH), creatine kinase (CK), blood urea nitrogen (BUN) and blood lactic acid (BLA), and increased the glycogen storage of liver and gastrocnemius muscle. WOPs also markedly inhibited fatigue induced oxidative stress by increasing the activity of superoxide dismutase (SOD), glutathione peroxidase (GPX) and decreasing the content malondialdehyde (MDA). Notably, WOPs improved the activity of pyruvate kinase (PK), succinate dehydrogenase (SDH), Na+-K+-ATPase, and enhanced the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content in skeletal muscles of mice. These results suggest that WOPs have beneficial anti-fatigue effects, which may be attributed to their positive effects on increasing glycogen storage, improving energy metabolism, inhibiting oxidative stress, enhancing mitochondrial function in skeletal muscle, and ameliorating the cell damage and the muscular injury.

In Vitro Antioxidant Activities and in Vivo Anti-Hypoxic Activity of the Edible Mushroom Agaricus bisporus (Lange) Sing. Chaidam.

With the rising awareness of a healthy lifestyle, natural functional foods have gained much interest as promising alternatives to synthetic functional drugs. Recently, wild Agaricus bisporus (Lange) Sing. Chaidam has been found and artificially cultivated for its thick fresh body and excellent taste, with its antioxidant and anti-hypoxic abilities unknown. In this work, the antioxidant potential of its methanolic, 55% ethanolic, aqueous extracts and crude polysaccharide was evaluated in different systems. The results showed that polysaccharide was the most effective in scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals, metal chelating activity and reducing power, with EC50 values of 0.02, 2.79, 1.29, and 1.82 mg/mL, respectively. Therefore, we further studied the anti-hypoxic activity of crude polysaccharide. The results turned out that polysaccharide (300 mg/kg) prolonged the survival time, decreased the blood urea nitrogen and lactic acid content as well as increased the liver glycogen significantly, compared with the blank control and the commercialized product Hongjingtian (p < 0.05). With such excellent activities, we purified the polysaccharide and analyzed its molecular weight (120 kDa) as well as monosaccharide components (glucose, fructose and mannose). This study indicated that wild Agaricus bisporus (Lange) Sing. Chaidam had strong potential to be exploited as an effective natural functional food to relieve oxidative and hypoxia stresses.

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens, Phlebia radiata.

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg(-1)). Combined with the data of laccase and manganese peroxidase, oxalate and H2O2 were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6-114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H2O2 and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.

Applications and mechanisms of ionic liquids in whole-cell biotransformation.

Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs' interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed.

The synergistic effect on production of lignin-modifying enzymes through submerged co-cultivation of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora using agricultural residues.

The lignin-modifying enzymes (LMEs) play an important role in decomposition of agricultural residues, which contain a certain amount of lignin. In this study, the production of LMEs by three co-cultivated combinations of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora and the respective monocultures was comparatively investigated. Laccase and manganese peroxidases (MnP) were significantly promoted in the co-culture of P. radiata and D. squalens, and corncob was verified to be beneficial for laccase and MnP production. Moreover, laccase production by co-culture of P. radiata and D. squalens with high ratio of glucose to nitrogen was higher than low ratio under carbon- and nitrogen-meager conditions. New laccase isoenzymes measured by Native-PAGE were stimulated by co-cultured P. radiata with D. squalens or C. subvermispora, respectively, growing in the defined medium containing corncob, but the expression of laccase was greatly restrained by the co-culturing of D. squalens with C. subvermispora. This study showed that the synergistic and depressing effects of co-cultivation of P. radiata, D. squalens and C. subvermispora on LMEs were species specific.

Effect of submerged culture conditions on exopolysaccharides production by Armillaria luteo-virens Sacc QH and kinetic modeling.

This work aimed to develop the submerged cultivation conditions for improved exopolysaccharides (EPS) production by Armillaria luteo-virens Sacc. The effects of culture temperature, aeration rate, inoculum level, initial pH, and additives on EPS formation and mycelial growth are investigated. The aeration rate, initial pH, and inoculum level significantly affected EPS production under the submerged cultivation. The developed conditions were as follows: cultivation temperature 23 °C, initial pH 5.0, aeration rate 0.5 vvm, 0.5% Tween 80, inoculum level 5% (v/v), and shaking speed 120 r/min. Under the developed conditions, the highest EPS production was 13.01 g/L at 5 days culture time. EPS production was examined in a 5 L bioreactor, and an unstructured kinetic model for EPS formation was well developed. The verified investigations in the large-scale cultivation system showed that the developed models are able to predict the submerged cultivation process of EPS formation. Current results revealed that the submerged cultivation conditions can be utilized to control EPS production, and the unstructured models developed are suitable for explaining EPS production by A. luteo-virens Sacc QH in a large-scale cultivation bioreactor.

Effect of ultrasonic-ionic liquid pretreatment on the hydrolysis degree and antigenicity of enzymatic hydrolysates from whey protein.

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins.

Optimization of acidic extraction of astaxanthin from Phaffia rhodozyma.

Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition: concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 degrees C, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 microg/g and 1516.0 microg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination.

The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Ginseng (Panax ginseng Meyer) Oligopeptides Protect Against Binge Drinking-Induced Liver Damage through Inhibiting Oxidative Stress and Inflammation in Rats.

Panax ginseng C.A. Meyer (ginseng) is an edible and traditional medicinal herb, which is reported to have a wide range of biological activity and pharmaceutical properties. There were more studies on ginsenoside and polysaccharides, but fewer on ginseng oligopeptides (GOPs), which are small molecule oligopeptides extracted from ginseng. The present study was designed to investigate the effects and underlying mechanism of ginseng oligopeptide (GOPs) on binge drinking-induced alcohol damage in rats. Sprague Dawley rats were randomly assigned to six groups (n = 10), rats in normal control group and alcohol model group was administered distilled water; rats in four GOPs intervention groups (at a dose of 0.0625, 0.125, 0.25, 0.5 g/kg of body weight, respectively) were administered GOPs once a day for 30 days. Experiment rats were intragastrically administered ethanol at a one-time dose of 7 g/kg of body weight after 30 days. The liver injury was measured through traditional liver enzymes, inflammatory cytokines, expression of oxidative stress markers, and histopathological examination. We found that the GOPs treatment could significantly improve serum alanine aminotransferase and aspartate aminotransferase, plasma lipopolysaccharide, and inflammatory cytokine levels, as well as the oxidative stress markers that were altered by alcohol. Moreover, GOPs treatment inhibited the protein expression of toll-like receptor 4, and repressed the inhibitor kappa Bα and nuclear factor-κB p65 in the liver. These findings suggested that GOPs have a significant protective effect on binge drinking-induced liver injury, and the mechanism possibly mediated by the partial inhibition of lipopolysaccharide-toll-like receptor 4-nuclear factor-κB p65 signaling in the liver.

Ginseng oligopeptides protect against irradiation-induced immune dysfunction and intestinal injury.

Intestinal injury and immune dysfunction are commonly encountered after irradiation therapy. While the curative abilities of ginseng root have been reported in prior studies, there is little known regarding its role in immunoregulation of intestinal repairability in cancer patients treated with irradiation. Our current study aims to closely examine the protective effects of ginseng-derived small molecule oligopeptides (Panax ginseng C. A. Mey.) (GOP) against irradiation-induced immune dysfunction and subsequent intestinal injury, using in vitro and in vivo models. Expectedly, irradiation treatment resulted in increased intestinal permeability along with mucosal injury in both Caco-2 cells and mice, probably due to disruption of the intestinal epithelial barrier, leading to high plasma lipopolysaccharide (LPS) and pro-inflammatory cytokines levels. However, when the cells were treated with GOP, this led to diminished concentration of plasma LPS and cytokines (IL-1 and TNF-α), suggesting its dampening effect on inflammatory and oxidative stress, and potential role in restoring normal baseline intestinal permeability. Moreover, the Caco-2 cells treated with GOP showed high trans-epithelial electrical resistance (TEER) and low FITC-dextran paracellular permeability when compared to the control group. This could be explained by the higher levels of tight junction proteins (ZO-1 and Occludin) expression along with reduced expression of the apoptosis-related proteins (Bax and Caspase-3) noticed in the GOP-treated cells, highlighting its role in preserving intestinal permeability, through prevention of their degradation while maintaining normal levels of expression. Further confirmatory in vivo data showed that GOP-treated mice exhibited high concentrations of lymphocytes (CD3+, CD4+, CD8+) in the intestine, to rescue the irradiation-induced damage and restore baseline intestinal integrity. Therefore, we propose that GOP can be used as an adjuvant therapy to attenuate irradiation-induced immune dysfunction and intestinal injury in cancer patients.

Effect of cultivating conditions on alpha-galactosidase production by a novel Aspergillus foetidus ZU-G1 strain in solid-state fermentation.

The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of alpha-galactosidase production in SSF were 60% initial moisture of medium, 28 degrees C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0 approximately 6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum alpha-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410: optimization of cultivation conditions using response surface methodology.

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.

Studies on optimization of nitrogen sources for astaxanthin production by Phaffia rhodozyma.

Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH(4))(2)SO(4), KNO(3) and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH(4))(2)SO(4), 0.49 g/L KNO(3) and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.

Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion.

Traditionally used as a restorative medicine, ginseng (Panax ginseng Meyer) has been the most widely used and acclaimed herb in Chinese communities for thousands of years. To investigate the immune-modulating activity of ginseng oligopeptides (GOP), 420 healthy female BALB/c mice were intragastrically administered distilled water (control), whey protein (0.15 g per kg body weight (BW)), and GOP 0.0375, 0.075, 0.15, 0.3 and 0.6 g per kg BW for 30 days. Blood samples from mice were collected from the ophthalmic venous plexus and then sacrificed by cervical dislocation. Seven assays were conducted to determine the immunomodulatory effects of GOP on innate and adaptive immune responses, followed by flow cytometry to investigate spleen T lymphocyte sub-populations, multiplex sandwich immunoassays to investigate serum cytokine and immunoglobulin levels, and ELISA to investigate intestinally secreted immunoglobulin to study the mechanism of GOP affecting the immune system. Our results showed that GOP was able to enhance innate and adaptive immune responses in mice by improving cell-mediated and humoral immunity, macrophage phagocytosis capacity and NK cell activity. Notably, the use of GOP revealed a better immune-modulating activity compared to whey protein. We conclude that the immune-modulating activity might be due to the increased macrophage phagocytosis capacity and NK cell activity, and the enhancement of T and Th cells, as well as IL-2, IL-6 and IL-12 secretion and IgA, IgG1 and IgG2b production. These results indicate that GOP could be considered a good candidate that may improve immune functions if used as a dietary supplement, with a dosage that ranges from 0.3 to 0.6 g per kg BW.

Suppression of TNF-α and free radicals reduces systematic inflammatory and metabolic disorders: Radioprotective effects of ginseng oligopeptides on intestinal barrier function and antioxidant defense.

Irradiation therapy is markedly associated with intestinal injure and oxidant stress. This study aimed to investigate the effects of ginseng (Panax ginseng C.A. Mey.) oligopeptides (GOP) on irradiation-induced intestinal injury and antioxidant defense in mice. BALB/c mice (8 weeks old) were randomly divided into six groups: vehicle control, irradiation control (IR), IR+whey protein [0.30 g/kg body weight (BW)], IR+GOP 0.15 g/kg BW, IR+GOP 0.30 g/kg BW and IR+GOP 0.60 g/kg BW. Postirradiation 30-day survival trial, white blood cells count and bone marrow hematopoietic system damage were performed to identify the injury degree induced by irradiation. Then, histopathology analysis was observed and intestinal permeability in vivo was quantified with fluorescein isothiocyanate-dextran. The enzyme-linked immunosorbent assay was used to determine antioxidant ability, plasma inflammatory cytokines, diamine oxidase (DAO) and endotoxin (LPS) levels. The immunohistochemistry assay was used to analyze the expression levels of tight junction proteins. We found that GOP-treated mice exhibited lower concentrations of plasma LPS and DAO and decreased instructors of inflammatory and oxidative stress which were linked to the lower intestinal permeability and higher tight junction proteins expression. The blockage of GOP was linked with the reduction of TNF-α and free radicals. The 15-day pretreatment of GOP could exhibit radioprotective effects, and another 15-day posttreatment benefited the quick repair of irradiation-induced injury. We confirm that GOP would exhibit effective therapeutic value on attenuating irradiation-induced hematopoietic, gastrointestinal and oxidative injury in cancer patients.

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410.

The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows: elastolysis temperature 50 degrees C, elastase concentration 1 x 10(4) U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasing elastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.

Synthesis and paste properties of octenyl succinic anhydride modified early Indica rice starch.

Octenyl succinic anhydride (OSA) modified early Indica rice starch was prepared in aqueous slurry systems using response surface methodology. The paste properties of the OSA starch were also investigated. Results indicated that the suitable parameters for the preparation of OSA starch from early Indica rice starch were as follows: reaction period 4 h, reaction temperature 33.4 degrees C, pH of reaction system 8.4, concentration of starch slurry 36.8% (in proportion to water, w/w), amount of OSA 3% (in proportion to starch, w/w). The degree of substitution was 0.0188 and the reaction efficiency was 81.0%. The results of paste properties showed that with increased OSA modification, the starch derivatives had higher paste clarity, decreased retrogradation and better freeze-thaw stability.