The use of canola meal as a substrate for xylanase production by Trichoderma reesei

Summary Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from Solka-floc, xylan or glucose. The maximum xylanase activity obtained from canola meal was 210 IU/ml in 9–12 days. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities. This system was more than or equally efficient as that produced using Solka-floc in hydrolysing canola meal, corn cobs, corn and wheat brans, straw, and larchwood xylan to fermentable sugars. Offprint requests to: Z. Duvnjak     

Enzymatic decrease in the phenolic content of canola meal using concentrated aqueous or aqueous/hexane slurries

An enzymatic process to decrease the phenolic content in canola meal was investigated. The new method was based on the addition of an enzyme preparation from the white-rot fungus Trametes versicolor to concentrated meal-buffer slurries. This approach eliminated the extraction of the valuable meal components such as proteins and carbohydrates. Two systems were considered: (i) slurries with canola meal concentrations higher than 33% [w/v]; (ii) slurries with canola meal concentrations equal to or less than 12.5% [w/v] with n-hexane as the main component of the continuous phase. The concentration of sinapic acid esters decreased by 99% after a 1.5, 2 and 3 hour long treatment of the meal with an initial moisture content of 75% at 90°C, 70°C and 50°C, respectively. The process was carried out at temperaturs as high as 110°C. Both the enzyme and the moisture concentrations influenced the enzymatic process and their action was coupled. The concentration of oxygen strongly affected the process. The enzymatic process was able to be carried out in the presence of hexane as the main component of the continuous phase. The optimum temperature for such a process was 30–40°C, At 30°C, after 1 h of treatment, the meal phenolic content was decreased by 97%. The water uptake by the meal was diminished in the presence of hexane.     

Production of a Laccase and Decrease of the Phenolic Content in Canola Meal during the Growth of the Fungus Pleurotus ostreatus in Solid State Fermentation Processes

Solid state fermentation of canola meal was carried out with the fungus Pleurotus ostreatus DAOM 197961, which is a producer of laccase. The aim of this study was to examine the effects of moisture content, inoculum size, homogenisation of inoculum and particle size of canola meal on the growth of the fungus, the production of a laccase and the decrease of the content of sinapic acid esters (SAE) in a solid state process. The results showed that the optimum moisture content, which was varied in the media between 50% and 75%, for the growth and enzyme production was 60%. The initial rate of SAE content decrease was faster in the media with 70% and 75% moisture than in those with lower moisture levels. In the study of the effects of inoculum concentration in the range of 1.1 mg to 5.5 mg/g of the medium, it was found that larger amounts of biomass and enzyme were produced in the media with inoculum concentrations from 1.1 mg to 3.3 mg/g of the medium than in the media with a higher inoculum concentration. The final and approximately the same concentrations of SAE were reached at the same time regardless of the inoculum concentration. Considering that the fungus formed pellets under the conditions at which it was grown during the inoculum preparation, it was necessary to break them by homogenisation prior to their utilisation as an inoculum. The homogenisation was carried out during a period between 15s and 200s. Although higher biomass concentrations and enzyme activities were obtained in the media which were inoculated with the inoculum homogenised for 15s and 30s, the maximum enzyme activities and biomass concentrations were reached in the media inoculated with the inoculum, which was homogenised for 120s and 200s. The time of inoculum homogenisation did not influence the kinetics of the SAE decrease. When the effects of the particle size of canola meal on the process were studied, it was found that larger particles of the meal in the solid media were more favourable for the production of the biomass and enzyme, and for a faster decrease of the SAE content than those of smaller sizes. From the obtained results it can be concluded that the tested variables have a significant influence on the growth of the fungus Pleurotus ostreatus DAOM 197961, the production of laccase and the decrease of the SAE content in canola meal. The data could be useful for the development of a solid state process for the production of laccase and for the decrease of the phenolics content in canola meal.     

Reduction of phytic acid content in canola meal by Aspergillus ficuum in solid state fermentation process

Summary Solid state fermentation (SSF) of canola meal has been carried out to reduce its phytic acid content using Aspergillus ficuum NRRL 3135. In certain batches, a complete reduction of phytic acid content in canola meal was achieved in 48 h. A larger amount of biomass in the inoculum and older inoculum increased the rate of phytic acid hydrolysis. The optimum moisture content of the medium was found to be 67% for phytic acid hydrolysis in an SSF process. The substitution of water in the semi-solid medium with acetate buffer resulted in faster reduction of the phytic acid content. A 15% increase in the amount of protein after 120 h of incubation was observed in the treated meal. The crude phytase preparation extracted from the canola meal after it was treated in an SSF process was also used for reduction of the phytic acid content in new batches of canola meal both in semi-solid medium and in liquid medium. In the semi-solid medium, 58% of the phytic acid was hydrolysed at 45°C in 20 h, while 100% hydrolysis was recorded at 50°C in 12 h in the liquid medium. The SSF process seems to be beneficial for the upgrading of canola meal by reducing both its phytic acid content and increasing the amount of protein.Offprint requests to: Z. Duvnjak     

Kinetics of Enhanced Substrate Consumption and Endo-β-xylanase Production by a Mutant Derivative of Humicola lanuginosa in Solid-state Fermentation

Summary Industrial byproducts namely canola meal, rice bran, sunflower meal, and wheat straw were used as substrates for endo-xylanase production by Humicola lanuginosemutant TH1 through solid substrate fermentation. The enzyme was secreted extracellularly by both wild and mutant cultures. Rice bran supported the maximum production of endo-xylanase followed by wheat straw, canola meal and sunflower meal. The highest activity was achieved after 72 h of culture and the highest yields from the above substrates were 842, 840, 610 and 608 IU per g substrate consumed respectively. The highest productivity (281 IU flask⁻¹ h⁻¹ corresponding to 5620 l⁻¹ h^-1) of endo-xylanase by the mutant of H. lanuginosa was 1.6-fold more than that produced by the parental organism in solid-state fermentation of rice bran at 45 °C. Maximum specific activity (180 IU mg⁻¹ protein) and substrate consumption rates were significantly more than those reported by previous researchers on Humicola sp. The mutant possessed markedly low accompanying cellulase activity. Thermodynamic studies revealed that the mutant required significantly lower activation energy for enzyme production and higher for thermal inactivation which signified that the endogenous metabolic machinery of mutant cells exerted more protection against thermal inactivation during product formation than that needed by its parental cultures.     

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

Effect of cellulase,phytase and pectinase supplementation on growth performance and nutrient digestibility of rainbow trout (Oncorhynchus mykiss,Walbaum 1792) fry fed diets containing canola meal

This experiment was conducted to evaluate the effects of supplementing exogenous enzymes on growth, feed conversion ratio (FCR) and apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fry diets containing 32% canola meal. Five experimental diets (including a control diet containing no enzymes) were prepared as isonitrogenous (44% crude protein) and isocaloric (4000 kcal DE kg¹). The four other diets contained either cellulase, phytase, pectinase or an enzyme mix (a mixture of cellulase, phytase and pectinase in the same ratio). The feeding trial was conducted in triplicate for 12 weeks in 15 tanks (100‐L). At the beginning of the experiment 20 rainbow trout fry (initial weight 1.23 g) were stocked into each tank. Mean water temperature in the rearing tanks was 11°C and water flow in each tank was 6 L min^?1. At the end of the experiment the growth parameters and FCR displayed no significant differences in enzyme supplementation (P > 0.05). In addition, no differences were observed in dry matter, protein, or lipid digestibility with enzyme supplementation (P > 0.05). The results of this study showed that the addition of pectinase, phytase, cellulase or an enzyme mix to a diet containing 32% canola meal had no effect on growth, feed efficiency or dry matter, protein, or lipid digestibility in rainbow trout fry.     

Phytase production and decrease of phytic acid content in canola meal byAspergillus carbonarius in solid-state fermentation

Solid-state fermentation (SSF) usingAspergillus carbonarius with canola meal as a substrate showed that production of phytase was associated with growth; maximum activity was achieved after 72 h. Apparent 25% and 10% increases in the protein content of the canola meal were noticed after 48 h and 72 h, respectively but total carbohydrate concentration had fallen by 25% by the end of fermentation. The rate of decrease of phytic acid content was optimum with a moisture content between 53% and 60%; homogenization of the inoculum for 120 s led to the greatest biomass and lowest phytic acid content. Inoculation of sterile meal led to lower phytic acid contents than inoculation of non-sterile meal.The authors are with the Department of Chemical Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada     

Comparison of three methods for the determination of sinapic acid ester content in enzymatically treated canola meals

The enzymatic reduction of sinapic acid ester content in canola meal using polyphenol oxidase from the fungusT. versicolor was investigated. To determine the effectiveness of this new process, the results obtained using two spectrophotometric methods and an HPLC analytical method for assaying sinapic acid ester content in the treated and untreated meals were compared. It was found that all the methods gave practically the same results when the samples from untreated canola meals were analysed. However, both of the spectrophotometric methods overestimated the sinapic acid ester content in the enzymatically treated meal by 7%–20%, as compared to the results obtained using HPLC. It was found that the sensitivity limits for the spectrophotometric methods used for the determination of sinapic acid ester content in enzymatically treated canola meals were 2.67 g and 1.47 g phenolics/kg meal for the direct and chemical spectrophotometric methods respectively. A correlation between the results obtained using the spectrophotometric and HPLC methods is given. The enzymatic treatment resulted in a negligible amount of phenolics in the treated meal.     

Effects of expeller pressed camelina meal and/or canola meal on digestibility, performance and fatty acid composition of broiler chickens fed wheat-soybean meal-based diets

This experiment was conducted to compare the effects of graded levels of camelina meal and/or canola meal on digestibility, performance and fatty acid composition of broiler chickens. A total of 180-day-old male broiler chicks were randomly assigned to one of the six treatments. The control diet was based on wheat and soybean meal and contained 15% canola meal. The experimental diets contained 3%, 6%, 9%, 12% or 15% camelina meal added at the expense of canola meal. Chromic oxide (0.35%) was added to all diets as a digestibility marker. On the morning of day 22, birds were killed by cervical dislocation and their abdominal fat pad was obtained. The apparent total tract digestibility of dry matter and energy as well as nitrogen retention all declined linearly (p?相似文献   

Characteristics of phytase produced by Aspergillus carbonarius NRC 401121 in canola meal

Aspergillus carbonarius NRC 401121 phytase was produced on canola meal in a solid-state fermentation process. A K_m value of 0.345 mM and a v_max of 0.81 units were determined for sodium phytate. The optimum pH and temperature were 4.7 and 53°C, respectively. Activation of the enzyme occurred when it was preincubated at higher temperatures for a period of time. The energy of activation, the entropy and the enthalpy changes were evaluated to be 7,800 cal/mole, 74 cal/(mole · K) and 24,000 cal/mole for this enzyme, respectively. The effect of time and the extractant: solid state culture ratio upon the single step extraction of phytase from a solid-state culture were evaluated. Mathematical correlations which fit the experimental data reasonably well were proposed for some of the studied processes.     

A seed coat-specific promoter for canola

The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The β-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola.     

Phytic acid content reduction in canola meal by various microorganisms in a solid state fermentation process

Solid state fermentation of canola meal has been carried out for the reduction of its phytic acid content using the following microorganisms: Rhizopus oligosporus NRRL 2990, Aspergillus niger NRC 5765 and NRC 401 121, Aspergillus ficuum NRRL 3135 and a wild Saccharomyces cerevisiae strain. The results showed that all these microorganisms can be used for the reduction of the phytic acid content in the tested material. A. ficuum which completely hydrolyzed the phytic acid in 48 hours was the most efficient. Buffered systems, aeration and an increase in inoculum concentration caused faster and higher reduction of phytic acid content in canola meal.     

The effect of feeding expeller-pressed canola meal on growth performance and diet nutrient digestibility in weaned pigs

The effects of feeding increasing levels of expeller-pressed (EP) canola meal in substitution for soybean meal as an energy and amino acid source were evaluated in 240 weaned pigs with an initial body weight of 7.3 ± 0.6 kg. Five pelleted wheat-based diets containing 0, 50, 100, 150 or 200 g EP canola meal/kg were formulated to contain 10.0 MJ net energy (NE)/kg and 1.18 g standardised ileal digestible (SID) lysine/MJ NE and were fed for 4 wk starting 1 wk after weaning at 19 days of age. Expeller-pressed canola meal was added at the expense of soybean meal and the diets were balanced for NE using canola oil and for amino acids using crystalline lysine, methionine, threonine and tryptophan. Increasing inclusion of EP canola meal linearly reduced (P<0.001) the apparent total tract digestibility of energy, dry matter and crude protein and the digestible energy content of diets. From 0 to 28 days on trial, increasing inclusion of EP canola meal did not affect body weight gain, feed intake and feed efficiency. In conclusion, up to 200 g EP canola meal/kg can replace soybean meal in diets formulated to equal NE and SID amino acid content and fed to nursery pigs starting 1 wk after weaning without reducing growth performance.     

The effect of phosphate concentration on phytase production and the reduction of phytic acid content in canola meal by Aspergillus carbonarius during a solid-state fermentation process

The use of canola meal, an abundant side-product of canola oil processing in Canada, as animal feed is hampered by high phytic acid levels that reduce metal cation availability. Aspergillus carbonarius grows well in a solid canola meal medium, produces phytase and reduces the phytic acid content to zero. Inorganic phosphate addition at a concentration of 1 mg and 5 mg/110 g solid-state culture system results in better growth of the microorganism, higher rates and levels of phytase production, and faster reduction of phytic acid content. Phosphate concentrations of 50mg and 100 mg/110 g inoculated system had a negative effect affecting primarily the initial rates of biomass and phytase production and phytic acid content reduction. Models that predict biomass production (expressed as glucosamine content) and phytase, as well as the reduction of phytic acid content in the solid-state cultures supplemented with phosphate are reported. They fit the experimental results reasonably well (with a maximum deviation of 7%).     

Production of angiotensin I-converting enzyme inhibitory peptides from defatted canola meal

To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4L and protease M “Amano” were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC₅₀ values of canola protein hydrolysates ranged from 18.1 to 82.5 μg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r = 0.5916, p < 0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products.     

Stability of a polyphenol oxidase from the white-rot fungus Trametes versicolor in the presence of canola meal

The stability of a polyphenol oxidase (PPO) preparation from the white-rot fungus Trametes versicolor during a process for the enzymatic decrease of the phenolic content of commercial canola meal (CM) was investigated. The effects of temperature, pH, protein origin and concentration, and meal particles were considered. The results showed that the thermal stability of the enzyme preparation was significantly increased in the presence of CM. The half-life times for the enzyme preparation, pre-incubated with CM at 50, 60, 70 and 75°C, were 45, 10.5, 3.5 and 1.5 hours, respectively; this represents an increase in the thermal stability of the enzyme preparation of up to four times in the presence of CM compared to the stability in the absence of CM. This effect was caused by the protective actions of both the CM particles and CM proteins, with the former responsible for 90% of the observed effect. The thermal stability of the enzyme in the presence of CM, from which 20% of the extractable proteins was extracted, was 5% lower compared to the stability in the presence of untreated CM. Changes in pH level from 5.0 to 3.2 resulted in a loss of stability comparable to that observed when the pre-incubation temperature was increased from 50 to 70°C. A semi-empirical model describing the changes in the concentration of the active enzyme pre-incubated in the presence and absence of CM at various incubation temperatures was proposed. A very good agreement between the model and experimental data was obtained. The proposed model, together with a general set of model parameters, can be used as a tool for the optimization of a process for the upgrade of CM by enzymatically decreasing the meal's phenolic content.     

Effects of untreated and heat-treated canola presscake on milk yield and composition of dairy cows

Five primiparous and five multiparous Holstein cows were used in two Latin square design experiments to determine effects of feeding unheated and heated canola presscake on milk yield and composition, and milk fatty acid concentrations of lactating dairy cows. Five diets that differed in level and source of dietary fat were formulated: a low fat control diet with 30 g kg⁻¹ fat from tallow, an unheated canola presscake supplemented diet (50 g kg⁻¹ fat), a heated canola presscake supplemented diet (50 g kg⁻¹ fat), a high tallow plus unheated canola meal supplemented diet (50 g kg⁻¹ fat), and a high tallow plus heated canola meal supplemented diet (50 g kg⁻¹ fat). In sacco ruminal degradability of heated and unheated canola presscake was compared with that of heated and unheated canola meal in a randomized complete block design using two ruminally fistulated cows. Heat treatment reduced ruminal DM and CP degradability of canola presscake. Multiparous cows fed diets supplemented with heated or unheated canola presscake produced more milk than those fed diets containing similar levels of fat from tallow with heated or unheated canola meal, respectively. High levels of fat from any diet reduced milk fat percentage for cows of either parity. Feeding heated canola products increased milk and milk protein yields in primiparous cows only, but cows of both parities fed diets containing canola presscake produced milk with lower concentrations of C12:0, C14:0, and C16:0 fatty acids than cows fed the canola meal and tallow diets, although concentrations of C18:1 n-9 were unaffected by fat source or level. Feeding canola products to dairy cows can alter milk fatty acid profile, but only primiparous cows have increased productivity as a result of feeding heated, versus unheated, canola presscake.     

Genome‐wide association analyses reveal complex genetic architecture underlying natural variation for flowering time in canola

Optimum flowering time is the key to maximize canola production in order to meet global demand of vegetable oil, biodiesel and canola‐meal. We reveal extensive variation in flowering time across diverse genotypes of canola under field, glasshouse and controlled environmental conditions. We conduct a genome‐wide association study and identify 69 single nucleotide polymorphism (SNP) markers associated with flowering time, which are repeatedly detected across experiments. Several associated SNPs occur in clusters across the canola genome; seven of them were detected within 20 Kb regions of a priori candidate genes; FLOWERING LOCUS T, FRUITFUL, FLOWERING LOCUS C, CONSTANS, FRIGIDA, PHYTOCHROME B and an additional five SNPs were localized within 14 Kb of a previously identified quantitative trait loci for flowering time. Expression analyses showed that among FLC paralogs, BnFLC.A2 accounts for ~23% of natural variation in diverse accessions. Genome‐wide association analysis for FLC expression levels mapped not only BnFLC.C2 but also other loci that contribute to variation in FLC expression. In addition to revealing the complex genetic architecture of flowering time variation, we demonstrate that the identified SNPs can be modelled to predict flowering time in diverse canola germplasm accurately and hence are suitable for genomic selection of adaptative traits in canola improvement programmes.     

双低菜粕高水平替代饲料鱼粉对大黄鱼潜在风险的评估:生长、健康和营养价值

研究从生长、健康和营养价值方面评估了高水平的双低菜粕替代饲料鱼粉对大黄鱼潜在的危害。在鱼粉含量60%的基础饲料（FM）上按照质量分数用双低菜粕分别替代15%（CM15）、30%（CM30）、60%（CM60）和100%（CM100）的鱼粉,配制成5种实验饲料。每种饲料投喂5个网箱的大黄鱼[初重（135.38±1.02）g],即每个处理5个重复,进行12周的养殖实验。结果表明,当双低菜粕替代水平在15%和30%时,大黄鱼的生长及饲料系数并没有受到显著性的影响。然而,当替代水平高于30%时,大黄鱼的末重和特定生长率均显著降低,而饲料系数显著升高（P < 0.05）。当替代水平达到100%时,大黄鱼摄食率达到最高值而肥满度达到最低值（P < 0.05）。在组织形态方面,大黄鱼摄食双低菜粕替代的饲料后肠道绒毛的弯曲程度减少并且排列更加不规则,而肝细胞则呈现出圆形空泡状并伴随着细胞核的偏移。对大黄鱼骨骼进行X-射线扫描发现,摄食双低菜粕的大黄鱼椎体和头部出现了畸形。在营养价值方面,双低菜粕替代鱼粉并未显著影响大黄鱼背肌的脂肪含量、蛋白含量和氨基酸组成,然而脂肪酸组成受到了显著影响,即N-6系列脂肪酸含量显著升高,而DHA与EPA含量显著降低（P < 0.05）。根据欧洲食品安全局（EFSA）的相关标准,这些营养价值的变化并没有影响大黄鱼作为健康食品的功能。由此可见,高水平（60%和100%）的双低菜粕替代鱼粉对大黄鱼的负面影响主要表现为降低大黄鱼的生长性能、改变肠道和肝脏组织形态,以及影响大黄鱼的骨骼健康。然而,双低菜粕替代鱼粉养殖大黄鱼的肌肉仍然符合人类的膳食要求。因此,双低菜粕替代鱼粉并没有影响大黄鱼作为食用鱼的营养价值。