Effect of substrate and fermentation conditions on pectinase and cellulase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 548 in submerged (SmF) and solid state fermentation (SSF)

The present study deals with the optimization of substrate and fermentation conditions for the production of both pectinase and cellulase by Aspergillus niger NCIM 548 under same fermentation conditions in submerged fermentation (SmF) and solid state fementation (SSF) using a central composite face centered design of response surface methodology (RSM). As per statistical design, the optimum conditions for maximum production of pectinase (1.64 U/mL in SmF and 179.83 U/g in SSF) and cellulase (0.36 U/mL in SmF and 10.81 U/g in SSF) were, time 126 h, pH 4.6, and carbon source concentration 65 g/L in SmF and were time 156 h, pH 4.80, and moisture content 65% in SSF. The response surface modeling was applied effectively to optimize the production of both pectinase and cellulase by A. niger under same fermentation conditions to make the process cost-effective in both submerged and solid state fermentation using agro industrial wastes as substrate.     

Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

Partial characterization of lipases produced by a newly isolated Penicillium sp. in solid state and submerged fermentation: A comparative study

The aim of this research was the partial characterization of enzymatic extracts produced by a newly isolated Penicillium sp. in submerged (SmF) and solid state fermentation (SSF). The partial characterization of the crude enzymatic extract obtained by SSF and SmF systems showed optimum activity at pH 5.5 and 47 °C, and pH 7.0 and 37 °C, respectively (15.17 U/mL and 11.28 U/mL). The crude enzymatic extracts obtained by SmF and SSF presented the best stability at pH from 4.9 to 8.5 and temperature from 25 °C to 35 °C and pH 7.0 and 25 °C, respectively. These results confirm the interesting potential of SSF, because, besides the higher activities obtained in this system, the half-life time at 25 °C was higher than that observed for the lipase extract obtained in the SmF system.     

Conidial production by Penicillium nalgiovense for use as starter cultures in dry fermented sausages by solid state fermentation

Penicillium nalgiovense is the typical species used as starter culture on the casing of dry fermented sausages and it has been often produced by solid state fermentation (SSF). Soy beans, maize kernels and wheat bran (WB) at 50% humidity were tested as substrates in SSF for conidial production and viability in P. nalgiovense. Among them WB was the best substrate for conidial production and viability. Thus, WB proved to be a suitable and convenient choice for conidial production by P. nalgiovense in SSF. By analysing conidial production on different membranes in malt extract agar (MEA) and WB no differences were observed between different treatments for the same substrate. Therefore, the practical choice of the inert support seems to be filter paper. A natural substrate like WB together with membranes as support helped the production of viable conidia by SSF as starters for dry fermented sausages.     

Effect of supportive enzymes on chemical composition and viscosity of rye mashes obtained by the pressureless liberation of starch method and efficiency of their fermentation

Rye mashes obtained by the pressureless liberation of starch method were treated with supportive enzymes such as pullulanase (Promozyme 200L), endo-1,4-β-xylanase (Shearzyme 500L), cellulase (Cellustar), β-glucosidase (Novozyme) and bacillolysin (Bacillus neutral proteinase) (Neutrase 0.5L). Effects of this treatment on physicochemical properties of these mashes, the course and yield of alcoholic fermentation and concentration of by-products in raw spirits were determined. Treatment with the endo-1,4-β-xylanase and cellulase coupled with β-glucosidase caused the greatest decrease in viscosity of the mashes by 97–99% in relation to the reference mashes (1,051 mPa s). Mashes treated with pullulanase and endo-1,4-β-xylanase contained more reducing sugars and were characterized by the relatively high starch saccharification degree. All the applied enzymatic preparations, in particular Promozyme 200L, Shearzyme 500L and Neutrase 0.5L improved the dynamics and yield of fermentation of the rye mashes. Enzymes contained in the examined preparations had no significant impact on concentration of by-products in raw spirits     

Juice extraction from mango pulp using an enzymatic complex of Trichoderma sp. produced by solid-state fermentation

In this study, 2 Trichoderma strains (T-I and T-II) were evaluated for the production, by submerged (SmF) and solid state fermentation (SSF) of an enzymatic complex with the capacity to degrade the cell components of mango peels, using these as support (in SSF) and source of nutrients. Highest enzymatic activity (7,552.5 units of endo-glucanase/L) was found in T-II by SSF. Efficiency of this crude enzymatic extract in the extraction of mango juice was evaluated, improving the yield up to 79%, representing an alternative to give an added value of mango peels improving the yields of production of mango juice.     

绿色木霉在稻壳和麸皮混合基质上固态发酵生产纤维素酶的研究

以稻壳和麸皮的混合物为主要原料 ,采用绿色木霉 (Trichodermasp 3 2 942 )固态发酵生产纤维素酶。浅盘发酵实验表明 ,稻壳经 4%的NaOH溶液 40℃浸泡 2 4h后 ,以 30 %的质量比添加到麸皮中发酵 ,滤纸酶活和CMC酶活比未经处理时提高了 2 7 5 %和 2 5 1 %;最优发酵条件为 :培养温度 30℃ ,接种量 2 0 %,培养基初始含水量 45 %～ 60 %,此时滤纸酶活和CMC酶活可达 5 1 5IU/g(干物质 )和 38 46IU/g(干物质 )。     

Enhanced Oil Recovery by Pre-treatment of Mustard Seeds Using Crude Enzyme Extract Obtained from Mixed-Culture Solid-State Fermentation of Kinnow (Citrus reticulata) Waste and Wheat Bran

Solid-state fermentation (SSF) of kinnow (Citrus reticulata) waste supplemented with wheat bran was used for production of cellulase, protease and pectinase with individual and mixed cultures of Trichoderma reesei and Aspergillus niger. Mustard seeds were pre-treated with crude filtrate extract (CFE) obtained from A. niger and T. reesei independently, and combination of the two cultures, prior to solvent extraction. Mixed-culture fermentation resulted in higher enzyme activity for filter paper cellulase (FPU), carboxymethyl cellulase (CMCase), β-glucosidase, and exoploygalacturonase (exo-PG) in comparison to fermentation with individual cultures. This study indicated that pre-treatment using crude enzyme obtained with mixed cultures enhanced the oil recovery by 11% as compared to control where no enzyme was used. Mustard seeds pre-treated with CFE obtained from mixed cultures having ratio of 2:1:1 for exo-polygalacturonase, FPU, and protease resulted in highest oil recovery. About 7–10% more oil was recovered when mustard seeds were pre-treated with CFE obtained from individual cultures, compared with control. Enzymatic pre-treatment also improved some of the quality attributes of mustard oil. To the best of our knowledge, this is the first study where mixed-culture SSF was attempted to produce enzyme consortium for pre-treatment of mustard seeds for enhanced oil recovery.     

Techno-economic analysis of processes for Aspergillus carbonarius polygalacturonase production

A techno-economic analysis of submerged (SmF) and solid state fermentation (SSF) processes for Aspergillus carbonarius polygalacturonase production was performed to make an appropriate process selection. The downstream processing involved integrated membrane process (IMP) and alginate affinity precipitation (AAP). For a production scale of 30kL purified polygalacturonase concentrate per year, the total upstream cost of SmF was 14% lower than the SSF process. Downstream processing cost by IMP was 47% lower than AAP. The SmF-IMP process required a total capital investment that was 15-24% lower than the SmF-AAP and SSF-AAP processes. The corresponding unitary product cost was also lower by 24-36% in SmF-IMP process. Thus the SmF-IMP process proved to be very attractive from an economic point of view.     

Bostrycin production by agro-industrial residues and its potential for food processing

Bostrycin, a red antibacterial agent produced by Nigrospora sp. no. 407, is considered for meat processing. To optimize production, the culture conditions of submerged fermentation (SmF) and solid-state fermentation (SSF) were investigated. The optimal SmF conditions were a medium containing 1.0% cane molasses and incubation at 30 °C and 150 rpm for 6 days. In SSF, other than bostrycin, less pigment was produced and the optimal ratio of bagasse to water was 1:2 for 10 days. The production and recovery rate of bostrycin by SmF were 120 mg/L and 40%, respectively. Bostrycin exhibited thermostable, pH-dependent color change and dose-dependent antibacterial activity against Clostridium botulinum. Bostrycin-modified meat turned strong red for at least 24 h and could not be removed by washing; bostrycin maintained its antibacterial activity with a bacteriostasis rate of 91% on Staphylcoccus aureus. This is an easy and inexpensive means of acquiring bostrycin from molasses and sugarcane.     

Influence of the addition of lupin sourdough with different lactobacilli on dough properties and bread quality

The aim of this research was to study the effects of solid‐state fermentation (SSF) with Lactobacillus sakei, Pediococcus pentosaceus and P. acidilactici on lupine sourdough parameters and lupine sourdough influence on the physical dough properties and wheat bread quality. The results showed that lactic acid bacteria (LAB) significantly reduced the pH and increased total titratable acidity (TTA) of SSF lupine. The highest protease activity in lupine is excreted by L. sakei (187.1 ± 8.6 PU g^?1), and the highest amylase activity, by P. pentosaceus (155.8 ± 7.5 AU g^?1). Lupine sourdough has a significant effect on the rheological properties of doughs, which affect the baking characteristics of the final product. In conclusion, it can be said that L. sakei, P. pentosaceus and P. acidilactici could be used for lupine SSF, and the addition of up to the 10% SSF lupine products increases the wheat–lupine bread quality.     

STUDIES ON CARBOHYDRATE METABOLIZING ENZYMES PART XXII. THE β-GLUCANASE SYSTEM OF MALTED BARLEY

Extracts of malted barley have been shown by molecular sieve chromatography to contain at least five enzymes which hydrolyse β-glucosidic linkages. In order of decreasing molecular weight, these are two β-glucosidases, an endo-β-1,4-glucanase, an endo-barley-β-glucanase and an endo-β-1,3-glucanase. This last-named enzyme was purified about 60-fold and shown to be specific for substrates containing only β-1,3-glucosidic linkages. This enzyme was activated by calcium ions, and deactivated by EDTA, but was not affected by chloride ions or a sulphydryl reagent. The endo-β-1,4-glucanase, which was purified about 50-fold, hydrolysed glucans containing only β-1,4- or a mixture of β-1,3- and β-1,4-linkages. The two β-glucosidases were shown to be true glycosidases and not exo-β-glucanases. The changes which occur in the levels of the various enzymic activities during the malting process have been measured.     

Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain

Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited >85% mannanase activity at the concentration of 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.     

微生物β-1,4-内切木聚糖酶研究进展

木聚糖是除纤维素之后,自然界中第二丰富的可再生资源,结构十分复杂,需要木聚糖降解酶系的协同作用才能将其完全水解。在木聚糖降解酶系中,β-1,4-内切木聚糖酶是使木聚糖完全水解最关键的酶,主要来源于细菌、真菌、放线菌等微生物,应用领域广,主要表现在食品、饲料、制浆造纸等行业。该文综述了微生物β-1,4-内切木聚糖酶的来源、选育、酶学性质、分类、分子结构、分子生物学及应用等研究进展。     

Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid State Fermentation

Aspergillus niger was subjected to UV radiation and chemical mutagenesis to develop its hyper-producing mutants for enhanced citric acid production. Ethyl methane sulfonate (EMS) and Ethidium bromide (EB) were used for chemical mutagenesis of Aspergillus niger. UV, and chemically treated mutants of Aspergillus niger were identified by using 2-deoxy, D-glucose as selective marker. The selected mutants were cultured in solid state fermentation (SSF) of sugarcane molasses medium (10%) using corn cobs, banana stalk, sugarcane bagasse, wheat straw, and wheat bran as carrier substrates. After pH adjustment and sterilization, the triplicate flasks were inoculated with 5 mLof homogenous spore suspensions of selected mutants of A. niger and the flasks were subjected to SSF under still culture conditions. The mutant EB-3 (treated with 1 mg/mL ethidium bromide for 120 min) giving highest citric acid yield (64.2 mg/mL) in 72 h was selected as hyper-producing mutant. Citric acid production process using EB-3 mutant was then optimized to enhance citric acid production by the mutant in SSF. Aspergillus niger EB-3 mutant could produce 67.72 mg/mL citric acid in 72 h using banana stalks as support material under optimum conditions of pH (pH 6), incubation temperature (35°C) and inoculum size (5 mL) in SSF.     

碱预处理秸秆同步糖化发酵生产丁二酸

研究了碱预处理秸秆及用琥珀酸放线杆菌Actinobacillus sucinogenes同步糖化发酵秸秆生产丁二酸。结果表明:用1.0%NaOH溶液于120℃分别预处理玉米、小麦和水稻3种秸秆2 h,其木质素的脱除率、纤维素与半纤维素的总保留率均在85%以上。以3种碱预处理后的秸秆为原料,在补加纤维素酶与纤维二糖酶的条件下,A.sucinogenes F3-21摇瓶厌氧发酵72 h,产丁二酸浓度分别为30.74 g/L、24.98 g/L和26.57 g/L;在7 L罐中厌氧发酵72 h,丁二酸浓度分别达到40.21 g/L,30.06 g/L和39.07 g/L,每克预处理秸秆产丁二酸分别为0.50g、0.38 g和0.49 g。并用钙盐法对玉米秸秆同步糖化发酵液进行提取,得到纯度为99.98%的丁二酸结晶。说明了玉米、小麦和水稻3种秸杆为原料进行同步糖化发酵生产丁二酸的可行性。     

Studies on the performance of a new bioreactor for improving antioxidant potential of rice

In the present investigation, we studied the performance of a self-designed new bioreactor (NB) for the improvement of phenolics and antioxidant activity in rice koji and the results were compared with the conventional solid-state fermentation (SSF). The non-fermented (control) and fermented masses obtained from two fermenters were extracted with water, methanol and ethyl acetate. Interestingly, rice fermented in NB resulted in a higher yield of phenolics and DPPH (1-1 Diphenyl-2-picryl hydrazyl) scavenging activity in each of the extraction media as compared to SSF and control. This might be due to the higher titre values of β-glucosidase (62.7%) and α-amylase (40.7%) in the extraction media of NB compared to SSF. It was verified by in vitro assay for phenolic enrichment in presence of α-amylase and β-glucosidase using rice as the substrate. An interesting finding was that though polyphenol content was maximum in ethyl acetate fraction, fractions extracted with water showed the highest antioxidant activity under all fermentation conditions, presumably because of the different phenolic composition which was substantiated by studying the antioxidant values of the extracts with same phenolic content in the antioxidant assay system.     

Effects of enzymatic hydrolysis treatments on the physicochemical properties of beef bone extract using endo- and exoproteases

This study reported the effects of enzymatic hydrolysis treatments on the physiochemical properties of beef bone extract using endo- and exoproteases. Each enzyme hydrolysis kinetics were studied using Michaelis–Menten model, and the ideal E/S ratio obtained for Protamex^® (P), bromelain (B) and Flavourzyme^® (F) was found to be 1.10%, 1.60% and 4.70% w/w, respectively. Seven hydrolysates were produced from single (P, B, F), simultaneous (P + F, B + F) and sequential (P > F, B > F) treatments, where bone extract hydrolysed by Flavourzyme^® exhibited highest DH and proportion of low molecular weight (Mw) peptides (<5000 Da) in single treatment. When Flavourzyme^® was used with Protamex^® or bromelain in simultaneous or sequential treatments, no significant differences in Mw distribution, exposed SH content, SS content and viscosity were evident compared with Flavourzyme^® only. This indicated that without the addition of other enzymes, Flavourzyme^® was capable of increasing the proportion of low Mw peptides and reduce viscosity.     

KINETICS OF BIOETHANOL PRODUCTION FROM WHEAT MILLING BY-PRODUCTS

An overview of the potential application of wheat milling by‐products as substrate for bioethanol production is presented. In order to select a suitable microorganism, model fermentations were conducted using glucose and dry baker's yeast. The overall ethanol yield was nearly stable (ca. 0.35 g/g), independent of mash glucose concentration; mashes with 100 g glucose/L resulted in an overall ethanol productivity of 3.48 g/L·h. Slurries containing low‐grade wheat flour (LG) (100, 200 or 300 g/L) were used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis. Fermentation performance was evaluated based on ethanol concentration (P), productivity (Q_v), yield (Y_P/S), production rate (Q_p) and glucose consumption rate (Q_s). Mashes containing 200 g LG/L produced about 52 g ethanol/L, with Q_vof 2.17 g/L·h. Based on the relatively high fermentation rate of LG, reaching peak ethanol productivity within ca. 9 h of SSF, considerable savings on fermentation time was achieved. Using Z. mobilis for LG fermentation, P was about 30% higher than that obtained with Saccharomyces cerevisiae.     

THE DEVELOPMENT OF β-D-GLUCANASES DURING THE GERMINATION OF BARLEY AND THE EFFECT OF KILNING ON INDIVIDUAL ISOENZYMES†

Two endo-1,3;1,4-β-D-glucanase isoenzymes developed in response to gibberellic acid, during the germination of barley. Two endo-1, 3-β-D-glucanases, one present in ungerminated, steeped grain, also developed but did not appear to be markedly stimulated by the hormone. A comparison of crude and partially purified malt extracts highlighted the errors that are involved in the specific determination of endo-1, 3;1, 4-β-glucanase activity in crude extracts. The development and effect of kilning on individual malt isoenzymes was demonstrated by carboxymethylcellulose (CM-cellulose) chromatography profiles. Kilning and dry-milling of germinated barley caused losses of 80–90% in the specific endo-1,3;1,4-glucanase activity. The effect was less pronounced if wet-milling was substituted for dry-milling. Extraction studies and CM-cellulose chromatography profiles indicated that both endo-1,3;1, 4-β-glucanase isoenzymes were heat labile and were particularly susceptible to oxidation. In contrast, endo-1,3-β-glucanase activity and cellobiase activity in malt extracts were less affected by the kilning process or extraction procedures. Preliminary results suggested that one of the endo-1,3-β-glucanase isoenzymes was more sensitive to kilning.