Sucrose Biotransformation to Fructooligosaccharides by <Emphasis Type="Italic">Aspergillus</Emphasis> sp. N74 Free Cells

Fructooligosaccharide production with the fructosyltransferase from free cells of the native strain Aspergillus sp. N74 at laboratory level was evaluated. The biomass of the native strain Aspergillus sp. N74 was produced in a sucrose fermentation medium and was employed in the enzymatic reaction in solutions of sucrose and phosphate buffer, where pH, temperature, and initial sucrose concentration effect were evaluated. Fructooligosaccharides and reaction subproducts were identified and quantified by high-performance liquid chromatography. The enzyme produced by the strain Aspergillus sp. N74 possessed hydrolytic and transfructosylating activities that changed with process conditions. The best transfructosylating condition was obtained at 80 min reaction time at pH 5.5, 60 °C, and initial sucrose concentrations higher than 550 g L⁻¹, with fructooligosaccharide production of about 50% w/w (based on initial sucrose concentration) and conversion selectivity higher than 90%. In addition, the transfructosylating and hydrolytic activities ratio was of 20. The high transfructosylating activity showed by the fructosyltransferase produced from the native strain Aspergillus sp. N74 suggest considering it as an alternative for the scale-up production of fructooligosaccharides by means of the whole microorganism at bench and pilot plant levels.     

<Emphasis Type="Italic">α</Emphasis>-Galactosidases production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-1

Extracellular and intracellular α-galactosidases were produced by yeast Debaryomyces hansenii UFV-1 grown on different media with several carbon sources. D. hansenii grown in YP-medium (1% yeast extract and 2% peptone) presented maximum cell mass (8.45 mg/mL) after 36 h of cultivation, with lactose as carbon source, followed by sucrose, glucose, raffinose, and galactose. Higher extracellular and intracellular α-galactosidases activities were observed at 48 h of D. hansenii cultivation in YPmedium containing galactose (0.97 and 5.27 U/mL) and lactose (1.28 and 4.88 U/mL), supporting the evidence for the model of induction for the yeast GAL/MEL regulon, such as described in Sacharomyces cereviseae.     

曲霉T3-5-1产单宁酶培养条件优化

以通过60Co 诱变得到的曲霉T3-5-1 为实验菌株,通过单因素与正交试验,对其产单宁酶的条件进行研究。结果表明：以β- 环糊精为碳源、蛋白胨为氮源、培养基初始pH 值为5.5、单宁酸质量分数为3.5%、接菌量为5%、摇瓶培养转速120r/min、250mL 三角锥瓶中装液50mL 培养基、培养温度30℃的培养条件为曲霉T3-5-1 的最佳产单宁酶条件。在此条件下,曲霉T3-5-1 产单宁酶比活力达到202.16U/mL,比优化前提高约4 倍。     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Production of gluconic acid from whey by free and immobilized Aspergillus niger

Deproteinized whey has been used as a fermentation medium for the production of gluconic acid by Aspergillus niger immobilized in polyurethane foam or free cells. Addition of a small amount of glucose (0.5%, w/v) in the whey medium enhanced the production of gluconic acid by 140% over the unsupplemented medium. Immobilized mycelia produced 92 g of gluconic acid from 1 L of whey medium containing 9.5% lactose and 0.5% glucose against 69 g by free mycelia. Immobilized mycelia can be reused.     

以柑橘果渣为主要原料生产细菌纤维素的研究

目的:拓宽细菌纤维素(BC)生产的原料来源,明确柑橘渣水对汉逊氏葡糖酸醋杆菌CIs51发酵产BC的影响。方法:以筛选自酸败柑橘果实的汉逊氏葡糖酸醋杆菌CIs51为发酵菌株,以柑橘果渣为主要原料,用扫描电镜观察其微结构,研究果渣预处理工艺、营养源、生长因子等对BC产量及微结构的影响。结果:柑橘果渣与水以1:8(W/V)的比例混合打浆,以150U/mL的果胶酶复配100U/mL纤维素酶,45℃水解2h;过滤后进行酵母前发酵工艺;选择蔗糖为碳源,添加量30g/L,(NH4)2SO4为氮源,添加量3g/L,酵母粉和柠檬酸的添加量分别为5g/L和1g/L。在此优化培养基中CIs51的产量达7.23g/L,明显高于其在HS培养基、柑橘渣水改良HS(CMHS)培养基中的产量(依次为4.02g/L及6.57g/L)。结论:柑橘渣水能够显著提高CIs51的BC产量,所得BC膜呈半透明质地,柔韧性好,具有替代椰子水进行工业化生产的前景。     

果胶酯酶的发酵培养基及培养条件优化

为提高果胶酯酶的产量,以果胶酯酶活力与生物量为指标,对塔宾曲霉CICC 2651产果胶酯酶的发酵培养基和培养条件进行了研究,通过单因素实验得到最优培养基和培养条件为:硫酸铵0.5%,果胶3%,Na₂SO₄ 0.04%,MgSO₄·7H₂O 0.04%,K₂HPO₄ 0.2%,培养温度30 ℃,初始pH为4.5,接种量5%,装液量40 mL。在优化工艺条件下,果胶酯酶活力达到1.53±0.09 U/mL,比初始条件提高了77.9%。通过发酵培养基和培养条件优化,塔宾曲霉CICC 2651发酵产果胶酯酶的能力大幅度提高。     

The effect of pectin concentration and degree of methyl-esterification on the in vitro bioaccessibility of β-carotene-enriched emulsions

Soluble fibers, like pectin, are known to influence the physicochemical processes during the digestion of dietary fat and may therefore affect the absorption of lipophilic micronutrients such as carotenoids. The objective of the current work was to investigate whether the pectin concentration and degree of methyl-esterification (DM) influence the bioaccessibility of carotenoids loaded in the oil phase of oil-in-water emulsions. The in vitro β-carotene bioaccessibility was determined for different oil-in-water emulsions in which 1 or 2% citrus pectin with a DM of 99%, 66% and 14% was present. Results show that pectin concentration and DM influence the initial emulsion properties. The most stable emulsions with the smallest oil droplets (D(v,0.9) of 15–16 μm) were obtained when medium or high methyl-esterified pectin was present in a 2% concentration while gel-like pectin structures (D(v,0.9) of 114 μm), entrapping oil droplets, were observed in the case where low methyl-esterified pectin was present in the aqueous emulsion phase. During in vitro stomach digestion, these gel-like structures, entrapping β-carotene loaded oil droplets, significantly enlarged (D(v,0.9) of 738 μm), whereas the emulsion structure could be preserved when the medium or high methyl-esterified pectin was present. Initial emulsion viscosity differences, due to pectin concentration and especially due to pectin DM, largely disappeared during in vitro digestion, but were still significant after the stomach digestion phase. The observed differences in emulsion structure before and during in vitro digestion only resulted in a significant difference between emulsions containing low methyl-esterified pectin (β-carotene bioaccessibility of 33–37%) and medium/high methyl-esterified pectin (β-carotene bioaccessibility of 56–62%).     

Extraction and partial characterization of Solanum lycocarpum pectin

In this work Solanum lycocarpum fruits were used as a source of pectin. The parameters of pectin extraction were examined using a 2³ factorial design, with temperature, pH and extraction time as independent variables. The extracted S. lycocarpum pectin and dried pulp were analyzed for the presence of antinutritional compounds, such as amylase and trypsin inhibitors, hemagglutinin, tannins, saponins, alkaloids and phytate. Pectin was characterized by measuring its methoxylation degree, intrinsic viscosity and molecular weight. The best pectin extraction conditions yielded as much as 33.6% (dry matter). The S. lycocarpum pectin was determined to be highly methoxylated (77.15%), and it showed an intrinsic viscosity 4.6% lower than that of citrus pectin, probably due to its lower molecular weight (177.76 kDa versus 224.48 kDa of that of citrus pectin). Although there were tannins and phytate in the dried pulp, the pectin fraction was free of these compounds.     

三株产几丁质酶菌株的筛选、产酶条件优化及水解虾壳的研究

目的:筛选鉴定产几丁质酶的菌株并优化其发酵条件,最终应用于虾壳降解研究。方法:以盐城市滩涂海泥为样品,利用平板筛选水解几丁质的菌株,运用生物信息学方法鉴定菌株,通过单因素优化其发酵条件,并将筛选得到的菌株和优化后的发酵条件用于虾壳发酵。结果:鉴定得到三株显著降解胶体几丁质的菌,分别是发光杆菌（Photobacterium sp. LYM-1）、需钠弧菌（Vibrio sp. WM-1）和希瓦氏菌（Shewanella sp. ZXY-1）;优化发酵条件:发光杆菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为32 ℃,发酵1 d酶活最高为15.37±0.55 U/mL,是优化前的4.37倍;需钠弧菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为7.5,温度为22 ℃,发酵2 d酶活最高为40.82±6.03 U/mL,是优化前的1.60倍;希瓦氏菌的碳源为几丁质10 g/L,氮源为(NH₄)₂SO₄ 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为22 ℃,发酵1 d酶活最高为25.64±3.29 U/mL,是优化前的2.47倍;三株菌均能利用虾壳产几丁质酶,但利用效率均低于几丁质,酶活力分别为10.25±0.95、32.16±2.25和21.81±4.27 U/mL。结论:本研究从盐碱地筛选得到三株产几丁质酶的菌株,优化后酶活力均得到提高,且均能利用虾壳产几丁质酶,为发酵虾壳制备几丁质酶提供新的菌株来源。     

Husk Tomato (Physalis ixocarpa Brot.) Waste as a Promising Source of Pectin: Extraction and Physicochemical Characterization

Husk tomato (Physalis ixocarpa Brot. var. Rendidora) waste was evaluated as a source of specialized pectin, and pectin extracted from this waste was characterized physicochemically. Fruit was blanched for 10 or 15 min and extracted in 0.1 N HCl for 15 to 25 min. Extracted pectin was subjected to physicochemical analysis. For all extraction conditions, the percentage of anhydrogalacturonic acid exceeded 60%, indicating that husk tomato was a good source of pectin. The degree of esterification of pectin molecules was 63% to 91%. The amount of extracted pectin decreased with increasing extraction time. The apparent viscosity of husk tomato pectin showed the characteristic behavior of pseudoplastic fluids. Neutral sugars were identified, and the amounts of 6 sugars (fucose, rhamnose, arabinose, galactose, glucose, and xylose) were quantified. Sugars identified in husk tomato pectin and present in the Rhamnogalacturonan I region, arabinose, galactose, and rhamnose suggest a highly branched structure, which will influence its future applications. Molecular weight values were 542 to 699 kDa, exceeding molecular weight values reported for commercial citrus pectins from 134 to 480 kDa. The extraction process significantly (P < 0.05) influenced the physicochemical properties of pectin. Up to 19.8% from the total amount of pectin in the husk tomato was extracted by 10 min of blanching and 20 min of a more heat treatment. Our findings indicate that husk tomato can be a good alternative source of pectin having highly distinctive physicochemical characteristics.     

柑橘果渣固态发酵产聚半乳糖醛酸酶

通过固态发酵,实现黑曲霉（Aspergillus niger）发酵生产聚半乳糖醛酸酶（PG）。以葡萄渣、苹果渣和柑橘渣为底物,利用黑曲霉18-23发酵生产聚半乳糖醛酸酶,考察不同果渣底物、可溶性碳源对胞外聚半乳糖醛酸酶合成的影响,并对该酶酶学性质进行研究。结果表明,柑橘渣最适合聚半乳糖醛酸酶的生产,苹果渣次之,二者的PG酶活力分别为56.9 U/g和42.1 U/g。乳糖对聚半乳糖醛酸酶的合成有诱导作用,可促进酶活力的增加,而槐糖无诱导作用,葡萄糖会抑制聚半乳糖醛酸酶的合成。聚半乳糖醛酸酶最适温度为50 ℃,最适pH值为5.5,且在温度40～50 ℃、pH 4.0～5.5稳定性良好,表明该酶在食品工业中具有潜在的应用价值。     

Xanthan Production by Mutant Strain of Xanthomonas campestris TISTR 840 in Raw Cassava Starch Medium

Raw cassava starch was used as carbon source in growing Xanthomonas campestris TISTR 840 for xanthan production due to its cheap price and mass production in Thailand. However, xanthan production with raw cassava starch yielded low content (4.31 g/l). A low level of amylase was detected in the XOL medium of X. campestris when raw cassava starch was used. Treatment of X. campestris TISTR 840 with ethyl methansulfonate resulted in the isolation of Xc-M, a strain that showed highest amylase overproduction. When cultured in bioreactor with a medium containing raw cassava starch, the growth of Xc-M cells was significantly higher than that of the wild type. The mutant produced 3.46- and 1.39-fold increased amylase activity and xanthan yield, respectively. Xc-M is useful for xanthan production in media containing raw starch as a carbon source.     

Evaluation of novel lactose-positive and exopolysaccharide-producing strain of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> for fermented foods

A novel strain of lactic acid bacteria Pediococcus pentosaceus P 773 was isolated from spoiled beer and identified by means of 16S rDNA sequence analysis. The ability to assimilate lactose as a sole carbon source as a specific feature for this strain was detected and confirmed on dairy substrates. In the presence of sucrose containing substrates (sucrose, raffinose) this P. pentosaceus P 773 lactose-positive strain produced a complex of extracellular polysaccharides (Qp = 0.08 g/l/h) with a molecular mass about 2,000 kDa composed by glucose and fructose residues at a ratio 3:1, respectively. These exopolysaccharides were capable to stimulate the growth rate and biomass productivity of common constituent cultures of probiotic dairy starters (Bifidobacterium lactis, Lactobacillus acidophilus, Streptococcus thermophilus) as well as were assimilated as a sole carbon source by these strains. The present study confirmed the presence of lactose-positive and exopolysaccharide-producing strain of P. pentosaceus in natural environment which could be used as a starter culture to impart more functional attributes to fermented food.     

Optimization of Chitosanase Production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> sp. Under Solid-State Fermentation

This study aimed at the optimization of the production of chitosanase in solid culture. Trichoderma koningii sp., an entomopathogenic fungus, was used to produce chitosanase under solid-state fermentation using a mixture of wheat bran and chitosan. The incubation period; addition of moistening water and culture medium composition were optimized. The protocol to extract the enzyme was also optimized. The optimal conditions for chitosanase production by T. koningii were obtained using a mixture of 3.0 g of wheat bran and 1.5 g of chitosan, with the addition of 2.5 mL of moistening water (pH 5.5) and of 2.5 mL of saline solution (pH 5.5) containing NaNO₃ (1.0 g/L), (NH₄)₂HPO₄ (1.0 g/L), MgSO₄.7H₂O (1.0 g/L), and NaCl (1.0 g/L). Optimal enzyme extraction was carried out adding 20 mL of sodium acetate buffer (200 mM, pH 5.5) at 30 °C under orbital agitation at 150 rpm for 6 min. The optimized production yielded 4.84 IU/gds.     

Gelatinised and hydrolysed corn starch is a cost-effective carbon source with higher production of L-lactic acid by Bacillus coagulans compared with glucose

L-lactic acid is an important organic acid widely used in pharmaceutical, food and textile industries. Bacillus coagulans BCS13002 can efficiently produce L-lactic acid with two kinds of carbon sources. BCS13002 produced L-lactic acid at a content of 10.23 ± 0.16 g/L and 11.67 ± 0.22 g/L, when glucose and gelatinised and hydrolysed corn starch （GHCS） were used, respectively. GHCS exhibits several advantages, including high yield of L-lactic acid and low cost. Proteomics analyses identified several key enzymes, which contributed to the higher production of L-lactic acid when GHCS was used as the carbon source. Those key enzymes were involved in the two-component system (SpoOF), pantothenate and CoA biosynthesis (pantothenate synthetase, 1.584-fold; dihydroxy-acid dehydratase, 1.517-fold), beta-alanine metabolism (1.605-fold) and valine, leucine and isoleucine biosynthesis (1.517-fold) pathways. This study provides a biological basis for using GHCS as a substitute of glucose in the production of L-lactic acid.     

Improving Solid-State Fermentation of Monascus purpureus on Agricultural Products for Pigment Production

In the present study, the effects of culture medium and temperature on red pigment production and mycelia growth were evaluated. The maximum red pigment production was found when Monascus purpureus CMU001 was cultivated on potato dextrose broth at 30 °C for 2 weeks. The highest amount of dry weight was achieved when cultivated on tryptone glucose yeast extract medium. Cheap agricultural products and residues were used as substrates for pigment production. Corn meal was the best substrate for pigment production (19.4 U/gds) when compared to peanut meal, coconut residue, and soybean meal. The highest pigment yield (129.63 U/gds) was found when corn meal was supplemented with 8% (w/w) glucose, followed by coconut residue (63.50 U/gds), peanut meal (52.50 U/gds), and soybean meal (22.50 U/gds). Galactose, sorbose, psicose, and mannitol were found to be good supplements next to glucose but not xylitol. Pigment was not stable at high temperature and long exposure to UV. The intensity of red pigment decayed 30.57% and 5.41% after autoclaving and pasteurization, respectively.     

盾叶薯蓣皂苷水解酶发酵条件优化研究

从盾叶薯蓣根茎清洗液中,筛选出一株产薯蓣皂苷水解酶菌株Aspergillus sp.,对该菌株产酶发酵条件进行优化研究。单因素试验结果表明,0.3%葡萄糖为碳源、0.4%蛋白胨为氮源,薯蓣皂苷水解酶活力为0.78 U/mL。正交试验结果表明,蛋白胨含量对酶活力的影响较大,产酶发酵培养基最佳配方为0.5%葡萄糖,0.6%蛋白胨,0.2%薯蓣皂苷,0.1%K2HPO4,0.05%MgSO4·7H2O,0.05%KCl,0.001% FeSO4·7H2O。在此最佳培养基配方条件下,薯蓣皂苷水解酶活力为0.96 U/mL。0.2%麦冬、0.2%绞股蓝、0.2%薯蓣三种混合皂苷作为诱导物时,薯蓣皂苷水解酶活力最高,达1.26 U/mL。     

绿色木霉HZ012发酵生产几丁质酶的研究

为探讨绿色木霉发酵合成几丁质酶的最优化培养条件,测定了各种不同培养条件下绿色木霉发酵合成几丁质酶的活力。结果表明:用0.9%几丁质和3.0%葡萄糖作为双碳源时几丁质酶的产量最高达0.192U/mL,高于单独使用葡萄糖或几丁质作为碳源时的最高产量;以1.0%蛋白胨作为氮源时几丁质酶的最高产量高于用1.0%酵母膏、1.0%硫酸铵、1.0%牛肉膏和1.0%亚硝酸钠作为氮源时的最高产量;发酵液的最佳初始pH为5.0,最佳微量元素添加量为2mL/L;在上述最优化培养条件下培养44h时几丁质酶活力达到最高值0.248U/mL。该方法获得的几丁质酶活力与Kapat等人报道的用Trichodermaharzianum生产几丁质酶的结果相比,酶活提高33%以上。     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.