Isolation and Screening of Lipase-Producing Fungi with Hydrolytic Activity

Lipases are enzymes that can be secreted by several microorganisms, making interesting the biodiversity exploration for searching new microorganisms able to produce these enzymes. Many agro-industrial residues can be used as potential substrates for production of enzymes. The main objective of this work was the isolation and screening of microorganisms with potential to produce lipases. Among 24 fungi, five were selected as good lipase producers using tributyrin on agar plates and solid state fermentation of soybean bran. Two of them were isolated from soil samples, another two from soybean bran, and one from dairy products. These fungi were identified by microcultivation technique as from Penicillium and Aspergillus genera. Through random amplified polymorphic DNA technique, the most promising strains could be genetically discriminated, selecting two fungi as good lipase producers but genetically different. One isolated from soybean bran could hydrolyze efficiently triglycerides with fatty acids with different chain length. Another isolated from dairy products was only effective to hydrolyze triglycerides with long-chain fatty acids. Two distinct groups could be verified by means of this technique, comprising the most productive strains and the lowest or nonproductive ones in terms of hydrolytic activity.     

Monoterpene bioconversion for the production of aroma compounds by fungi isolated from Brazilian fruits

This work aimed to isolate fungal strains from Brazilian fruits and select potential biocatalysts for the bioconversion of the monoterpenes citronellol, limonene, linalool, and geraniol. Among the 36 fungal strains isolated, 12 were capable to convert citronellol into rose oxide and its isomers, while the biotransformation of limonene led to the formation of 2 derivatives of high market value, carvone and α-terpineol. Furthermore, geraniol was converted into 2 new products and linalool into linalool oxide, ocimenol, geraniol, and also α-terpineol by using several strains. Accordingly, the microorganisms LB-2025, LB-2036, and LB-2038 were selected for further experiments and identified as Penicillium sp., Penicillium sp., and Aspergillus sp., respectively. Induction studies with substrates and products have been evaluated and the production of rose oxide was enhanced from 5- to 8-fold, while the products originated from the biotransformation of limonene have not changed. Thus, this work demonstrates the biotechnological production of new flavor compounds.     

Tempering–preservation treatment inactivated lipase in wheat bran and retained phenolic compounds

Wheat bran is rich in functional ingredients, but the high level of lipase limits its applications. Tempering–preservation treatment (at 70–90 °C with moisture of 20%–40% for 1–4 h) was exploited for stabilising wheat bran and its effect on polyphenols was investigated. The results showed that more lipase was inactivated at higher tempering moisture, temperature and longer time. The optimum condition for inactivation of wheat bran lipase was 30% moisture and 90 °C for 4 h. The inactivation rate reached 93.8% with a residual enzyme activity of 0.264 U g⁻¹. Under the optimum condition, the sum of free phenolic acids rose from 25.4 to 55.8 µg g⁻¹. As for bound phenolic acids, there was a slight increase of hydroxybenzoic acid derivatives but a slight decrease of hydroxycinnamic acid derivatives. The total contents of phenolic acids before and after stabilisation were not significantly different. This study showed the possibility of using tempering–preservation as an efficient method for inactivation of wheat bran lipase while maintaining its phenolic compounds, which could be used in the production of whole wheat flour.     

Microbiological studies on cassava fermentation for ‘lafun’ production

The microorganisms involved in the traditional fermentation of cassava for ‘lafun’ production were investigated. The microorganisms isolated during the fermentation include Bacillus sp., Leuconostoc sp., Klebsiella sp., Corynebacterium sp., Lactobacillus sp., Candida sp., Aspergillus sp., and Geotrichum sp. The moulds disappeared within the first 36h of fermentation. Bacillus sp. which were present at the beginning of fermentation decreased drastically as fermentation progressed. The yeasts appeared between 24–48h of the fermentation and increased rapidly. The lactic acid bacteria were implicated throughout the duration of the fermentation. Bacillus sp., Corynebacterium sp., Candida sp. and the lactic acid bacteria were considered to play important roles. The titratable acidity of the steeping water and tubers increased rapidly reducing the pH to below 4·0. The temperature of the steeping water varied between 30–32°C and usually not above the ambient. The moisture content of the fermenting tubers increased from 65% to 70% within the first 36 h and was never below 60% during the steeping period. The post-fermentation sun-drying reduced the moisture content to 12–15%.     

Isolation and characterization of folate-producing bacteria from oat bran and rye flakes

The aim of this research was to identify endogenous bacteria in commercial oat bran and rye flake products in order to study their folate production capability while maintaining the soluble dietary fibre components in physiologically active, unhydrolyzed form.Fourty-two bacteria were isolated from three different oat bran products and 26 bacteria from one rye flake consumer product. The bacteria were tentatively identified by sequence analysis of the 16S rRNA genes. The identification results revealed up to 18 distinct bacterial species belonging to 13 genera in oat bran, and 11 species belonging to 10 genera in rye flakes. The most common bacterial genus in oat bran was Pantoea, followed by Acinetobacter, Bacillus, and Staphylococcus. Pantoea species dominated also in rye flakes. The extracellular enzymatic activities of the isolates were studied by substrate hydrolysis plate assays. Nearly 80% of the isolates hydrolyzed carboxymethylcellulose, whereas starch-degrading activities were surprisingly rare (10%). Beta-glucan was hydrolyzed by 19% of the isolates. Protease, lipase or xylanase activity was expressed by 24%, 29%, and 16%, respectively, of the isolates. Representatives of the genera Bacillus, Curtobacterium, Pedobacter, and Sanguibacter showed the highest diversity of enzymatic activities, whereas members of Janthinobacterium and Staphylococcus possessed no hydrolytic activities for the substrates studied. Production capability for total folates was analyzed from aerobic cell cultures at the stationary growth phase. The amount of folates was determined separately for the cell mass and the supernatant by microbiological assay. For comparison, folate production was also examined in a number of common lactic acid bacteria. The best producers in oat bran belonged to the genera Bacillus, Janthinobacterium, Pantoea, and Pseudomonas, and those in rye flakes to Chryseobacterium, Erwinia, Plantibacter, and Pseudomonas. Supernatant folate contents were high for Bacillus, Erwinia, Janthinobacterium, Pseudomonas, and Sanguibacter. Compared to the endogenous bacteria, lactic acid bacteria were poor folate producers. The results of this work provide the first insight into the potential role of endogenous microflora in modulating the nutrient levels of oat and rye based cereal products, and pave way to future innovations of nutritionally improved cereal foods.     

Improving storage property of wheat bran by steam explosion

Steam explosion (SE) was exploited for stabilizing wheat bran (WB), and its effects on nutrients retention of WB were investigated. Result showed that SE could effectively inactivate lipase and peroxidase of WB at 0.8 MPa (170 °C) for 5 min while lipid and protein were not loss. Total flavonoids, phenolics contents, soluble dietary fibre content and DPPH radical scavenging activity in WB after SE increased by 198%, 83%, 27% and 21% respectively compared with those after conventional thermal sterilization (0.1 MPa (121 °C) for 20 min), while fatty acid value and peroxide value and insoluble dietary fibre content deceased by 21%, 75% and 24%, respectively. SE inhibited hydrolysis rancidity of wheat bran and reconstituted whole wheat flour (wholemeal) in the accelerated storage test. Therefore, SE may be a new efficient technology that could stabilize cereal bran and further promote its products development.     

Optimization for rice koji preparation using aspergillus oryzae CJCM-4 isolated from a korean traditional meju

To obtain a useful mold strain in rice-doenjang production, enzyme activities were investigated in rice and wheat koji prepared with 13 different strains isolated from traditional Korean meju. The CJCM-4 strain, which had been selected as a superior strain for the production of protease, was identified as Aspergillus oryzae by the gene sequence of the ITS-5.8S rDNA region as well as aflatoxin production capacity. The enzyme activities of koji prepared with CJCM-4 according to the polishing degree of rice bran were examined. The enzyme activities of koji prepared with 70% milling degree of rice bran were higher than those of other kojis. Protease activity showed its highest level on day 3 at 30°C. Glucoamylase activity showed its highest level on day 2 at 30°C. The optimum enzyme production of koji was determined at different conditions such as incubation time, temperature, moisture, and inorganic salt content. The optimum ratio of water added to the rice for the production of enzymes was 35% on the dry basis of the rice, and the optimum temperature was 25°C. When inorganic salts were added to increase enzyme activity, the combined addition of 0.15% CaCO₃ and 0.05% NaH₂PO₄ resulted in the highest enzyme activities.     

Effects of enzymatic treatment conditions on dietary fibre content of wheat bran and use of cellulase-treated bran in cookie

A commercial cellulase preparation (Viscozyme Cassava C) was employed to process wheat bran to improve the ratio of insoluble to soluble dietary fibre. The effects of initial moisture content, enzyme dosage, and incubation time on the content of soluble and insoluble fibre during the enzymatic treatment were investigated. The appropriate conditions for the cellulase treatment were initial moisture content of 0.75 g water g⁻¹ dry matter, enzyme dosage of 9 U g⁻¹ dry matter and incubation time of 120 min under which the ratio of insoluble to soluble fibre of wheat bran was lowered by 42%. Untreated and cellulase-treated wheat bran was incorporated into cookies at levels of 0%, 20%, 30%, 40% and 50%. Cookie with 50% cellulase-treated bran showed 21% lower ratio of insoluble to soluble fibre as well as 14% lower hardness and 13% higher overall acceptability than sample with the same amount of untreated bran.     

Biochemical characterisation and application of lipases produced by Aspergillus sp. on solid‐state fermentation using three substrates

Lipase from Aspergillus sp. obtained by solid‐state fermentation (SSF) on wheat bran (LWB), soybean bran (LSB) and soybean bran combined with sugarcane bagasse (LSBBC) were 67.5, 58 and 57.3 U of crude lipase per gram substrate, respectively. The optimum pH of activity and stability of the LWB was between 8 and 9, and the optimum temperature of activity and stability was 50 °C and up to 60 °C, respectively. The LSB and LSBBC showed two peaks of optimum pH (4 and 6) and optimal values of temperature and stability at 50 °C. The LSB was stable in the pH range of 6–7, while LSBBC in the range of pH 4–7. All the enzymes show activities on p‐nitrophenyl esters (butyrate, laurate and palmitate). LWB stood out either on the hydrolysis of sunflower oil, presenting 66.1% of the activity over commercial lipase and on the esterification of oleic acid and ethanol, surpassing the activities of the commercial lipases studied. The thin layer chromatography showed that LWB and LSB have produced ethyl esters from corn oil, while LWB produced it from sunflower oil.     

热处理对米糠及米糠油特性的影响

用常压蒸煮、高压蒸煮和微波处理米糠,研究热处理方法对米糠及米糠油品质的影响。结果表明,三种热处理方式均能使米糠的水分下降,抑制过氧化物酶和脂肪酶活力,使储藏过程中脂肪酸值上升速度减慢。其中常压蒸煮米糠的过氧化物酶和脂肪酶活力下降最多;微波处理的米糠在储藏过程中脂肪降解最慢,过氧化物酶和脂肪酶活力有下降的趋势,提出的米糠油品质优于常压蒸煮和高压蒸煮。     

Rheological properties of wheat flour and quality characteristics of pan bread as modified by partial additions of wheat bran or whole grain wheat flour

The use of bran and whole grain flour changes dough rheology and causes difficulties in manufacturing bakery products. The aim of this study was to analyse the influence of substituting refined wheat flour (WF) by wheat bran (WB; 5%, 10%, 20%, 30% and 40%) or whole grain wheat flour (WGWF; 10%, 20%, 30%, 40% and 50%) on dough rheological properties and pan bread quality characteristics. The addition of WB and WGWF increased water absorption and resistance to extension and decreased stability, extensibility and peak viscosity. Effects with WB were more pronounced. The presence of WB or WGWF increased crumb moisture content, firmness and hardness and decreased specific volume of pan bread. It is important to set new farinographic and extensographic standards when using WB and WGWF, allowing for a correct correlation between rheology and quality characteristics of bakery products, as the same standards used for WF are not valid.     

红外加热米糠稳定化研究

研究以过氧化物酶残余活力和残余水分含量为评价指标,采用3因素3水平正交试验设计,研究了红外干燥中3个主要因素(温度60～80℃,处理时间15～25min,物料厚度20～30mm)对米糠稳定化效果的影响。结果表明:较佳处理工艺参数为:加热温度80℃,处理时间20min,物料厚度25mm。处理后的米糠过氧化物酶残余活力为3.91%,水分含量为4.24%。经过90d的贮存,红外处理后的米糠在室温下游离脂肪酸值(FFA)18.0mgKOH/g,4℃冷藏下10.1mgKOH/g,远远低于对照组(98.8mgKOH/g和56.9mgKOH/g)。说明红外处理对米糠稳定化效果非常显著,同时冷藏有助于有效抑制米糠酶的活性。强化储藏试验表明,90d后经稳定的米糠稳定性提高了5倍,充分说明红外处理能够使米糠长期稳定化。     

Stagnant Effective Thermal Conductivity of Agro-Industrial Residues for Solid-State Fermentation

The stagnant effective thermal conductivities (K₀) of sugar cane bagasse (SCB), wheat bran (WB), orange pulp and peel (OPP) and their combination (weight proportion 1:2:2 SCB/OPP/WB) were obtained using the line heat source method. These solid materials were applied to pectinase production via solid-state fermentation. The moisture content ranged from 4 to 80% (w.b.). A conduction mechanism through the porous media was observed, along with conduction through a liquid film and contact thermal resistance between the samples and the probe. K₀ was low for intermediate moisture contents and approached the molecular conductivity of water for high moisture contents.     

Effects of replacing pork back fat with vegetable oils and rice bran fiber on the quality of reduced-fat frankfurters

The effects of substituting olive, grape seed, corn, canola, or soybean oil and rice bran fiber on the chemical composition, cooking characteristics, fatty acid composition, and sensory properties of low-fat frankfurters were investigated. Ten percent of the total fat content of frankfurters with a total fat content of 30% (control) was partially replaced by one of the vegetable oils to reduce the pork fat content by 10%. The moisture and ash content of low-fat frankfurters with vegetable oil and rice bran fiber were all higher than the control (P < 0.05). Low-fat frankfurters had reduced-fat content, energy values, cholesterol and trans-fat levels, and increased pH, cooking yield and TBA values compared to the controls (P < 0.05). Low-fat frankfurters with reduced-fat content plus rice bran fiber had sensory properties similar to control frankfurters containing pork fat.     

Impact of transglutaminase treatment on properties and in vitro digestibility of white bean (Phaseolus vulgaris L.) flour

Common beans (Phaseolus vulgaris L.) are rich in nutrients and have significant amounts of proteins and complex carbohydrates, besides to be rich in unsaturated fatty acids and dietary fibres. Consumption of beans could be improved by processing them into flour. In this study the effect of microbial transglutaminase (TG) on the structure, physical (colour parameters, moisture, water holding capacity), thermal properties and in vitro digestion of undehulled (WB) and manually dehulled (SB) flour samples from white common beans (P. vulgaris L.) was evaluated. Flour samples were incubated in the absence and presence of TG (WB/TG and SB/TG). We observed that the enzyme is able to catalyse the formation of polymers, suggesting that the proteins occurring in the bean flour act as TG substrates. Microstructure of samples was examined by Scanning Electron Microscopy (SEM), while thermal properties were studied by Differential Scanning Calorimetry. Microstructural results showed that the TG-treated samples possess a more compact structure, made of starch granules surrounded by proteins that, presumably, contain TG-catalysed polymers. Moreover, TG treatment had a major impact on colour, water holding capacity (WHC) and thermal properties. In particular, WB and SB samples presented a darker colour than WB/TG and SB/TG samples, while the latter showed reduced WHC that was only 30% and 37% of WB and SB samples, respectively. The transition enthalpy (ΔH) in the temperature range from 57 to 70 °C (WB, WB/TG) and from 60 to 68 °C (SB, SB/TG) followed the order: WB/TG > WB and SB/TG > WB, respectively. In vitro digestion experiments indicate that the presence of isopeptide bonds decreased the digestibility of TG-treated flour samples.     

乳酸菌发酵米糠产γ-氨基丁酸最适条件的研究

使用乳酸菌发酵米糠,以菌种、菌种添加量、发酵温度、发酵时间为单因素研究其对米糠发酵液中γ-氨基丁酸(GABA)含量的影响,在最适条件下,采用混料设计法,使用混合乳酸菌发酵米糠,以发酵液中GABA含量为指标,确定乳酸菌发酵米糠的最优条件为:在混合菌种嗜热链球菌S1添加量为1.55%,保加利亚乳杆菌L1添加量为1.45%,50℃下发酵米糠14h,发酵液中GABA含量最高,为287.975mg/100g。     

Characterization of Microencapsulated Rosemary Essential Oil and Its Antimicrobial Effect on Fresh Dough

This study aimed to characterize rosemary essential oil particles obtained by spray-drying and to evaluate their antimicrobial activity. Measurements of mycelial growth of Penicillium and Aspergillus fungi, isolated from fresh dough, were made applying oil concentrations of 1.0, 5.0, 10.0, 20.0, and 50.0 μL/mL. Fungi and yeast counts in the fresh dough submitted to the control (no oil), pure oil, and microencapsulated oil treatments were also conducted. The microcapsules showed 50 % oil retention after drying, and the major oil components identified were 1,8-cineole (29.0 %), camphor (26.6 %), and α-pinene (10.6 %). The analysis of the particles revealed surfaces without fissures, with a mean particle size of 12.2 μm and presenting an amorphous structure. The growth inhibiting effect of Penicillium sp. fungus, compared to the control, was verified at concentrations of 1.0, 5.0, and 10.0 μL/mL rosemary essential oil, which did not differ among them. For Aspergillus sp., the application of 10.0 μL/mL oil provided greater inhibition compared to 1.0 and 5.0 μL/mL. Complete inhibition occurred with the application of 50.0 μL/mL for both fungal genera tested. At 8 days of dough storage at 25 °C, a decrease of at least 0.7 and 1.5 log cycles of fungal growth was observed in the dough with pure oil and that with microencapsulated oil, respectively, relative to the control. The microencapsulation process retained the antimicrobial property of rosemary essential oil and provided further extension of this activity over time when applied to fresh dough.     

Enzymatic polishing of rice - A new processing technology

A novel processing technology was developed to polish rice in a more selective way with the help of xylanase and cellulase enzymes. The enzymes are produced from Aspergillus sp. and Trichoderma sp., respectively. Brown rice was soaked in water at 35 °C for 24 h to attain a saturated moisture level of 35.5 g/100 g, which leads to an increase in GABA concentration to 27 mg/100 g of rice. Enzymatic treatment was done for 2 h at 50 °C. The enzymes acted upon the non-starch polysaccharides of the bran layers releasing their monomeric sugar constituents, as detected through HPLC. Enzyme degraded surface structure of the rice grain was also studied by Scanning Electron Microscopy. Crude oil and crude fibre content of brown rice was reduced by 16 g/100 g and 20 g/100 g, respectively, through enzymatic treatment over brown rice.     

Effect of antioxidants and competing mycoflora on Fusarium verticillioides and F. proliferatum populations and fumonisin production on maize grain

This study was carried out to determine the temporal effect of the antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) at doses of 500 and 1000 μg/g on the growth of Fusarium verticillioides and F. proliferatum inoculated on natural maize grain in the presence of the competing mycoflora and fumonisin production at 0.98 and 0.95 water activity (a_w) over a 28-day storage period. The reduction in the log colony forming units (CFU) of Penicillium, Aspergillus and Fusarium populations was 10-100 fold depending on dose of BHA or PP, a_w and time. However, the populations of all three groups were higher at 0.98 a_w than 0.95 a_w. BHA at 500 μg/g and 0.95 a_w reduced the fumonisin content by 82% after 7-14 days incubation, but at the end of the experimental period the reduction was only 32%. A higher reduction in the level of fumonisin produced (77%) was achieved with BHA at 1000 μg/g after 28 days. PP at 500 and 1000 μg/g decreased fumonisin production throughout the incubation period in the drier treatment, but at 0.98 a_w control of toxin production was only achieved after 7-14 days. The reduction in the fumonisin levels could be due to the combined effect of antioxidants, and the competing mycoflora, mainly Aspergillus and Penicillium species.     

Comparison of releasing bound phenolic acids from wheat bran by fermentation of three Aspergillus species

Wheat bran was fermented at 28 °C for 7 days under 70% humidity by Aspergillus niger, Aspergillus oryzae and Aspergillus awamori. Total phenolic content (TPC) of the unfermented sample was 1531.5 μg g^?1 wheat bran. After the fermentation of Aspergillus awamori, Aspergillus oryzae and Aspergillus niger, TPC reached 5362.1, 7462.6 and 10 707.5 μg g^?1, respectively. The antioxidant activity in the extractions of fermented wheat bran also increased significantly compared with the unfermented sample (P < 0.05). Aspergillus niger showed the greatest capacity to release bound ferulic acid (416.6 μg g^?1). Aspergillus oryzae and Aspergillus awamori had the advantages of releasing more chlorogenic acid (84.0 μg g^?1) and syringic acid (142.3 μg g^?1), respectively. The destructive effect of Aspergillus niger on wheat bran structure was the strongest, followed by that of Aspergillus oryzae. This effect of Aspergillus niger may be due to its higher cellulase, xylanase, arabinofuranosidase and β‐xylosidase activities. Besides, Aspergillus oryzae possessed higher β‐glucosidase activity, and Aspergillus awamori had higher α‐amylase and feruloyl esterase activities. Aspergillus niger may be the best to release bound phenolic acids in the three Aspergillus species. These will provide the helpful information for understanding mechanism of the fermentation by Aspergillus species releasing bound phenolic in wheat bran.