Novel techniques for the mass production of nutritionally improved, fungus-treated lignocellulosic biomass for ruminant nutrition.

BACKGROUND: Laboratory-scale experiments have shown that treatment with selective lignin-degrading white-rot fungi improves the nutritional value and ruminal degradability of lignocellulosic biomass (LCB). However, the lack of effective field-applicable pasteurization methods has long been recognized as a major obstacle for scaling up the technique for fungal treatment of large quantities of LCB for animal feeding. In this study, wheat straw (an LCB substrate) was subjected to four field-applicable pasteurization methods - hot-water, formaldehyde fumigation, steam, and hydrated lime - and cultured with Pleurotus ostreatus grain spawn for 10, 20, and 30 days under solid-state fermentation. Samples of untreated, pasteurized but non-inoculated and fungus-treated straws were analyzed for chemical composition, aflatoxin B1 (AFB1 ), and in vitro dry matter digestibility (IVDMD), in vitro total gas (IVGP), methane (CH4 ), and volatile fatty acid (VFA) production. RESULTS: During the 30-day fungal treatment, steam and lime pasteurized straws had the greatest loss of lignin, resulting in marked improvements in crude protein (CP), IVDMD, IVGP, and total VFAs. Irrespective of the pasteurization method, the increase in IVDMD during fungal treatment was linearly (R2 = 0.77-0.92) related to lignin-loss in the substrate during fungal treatment. The CH4 production of the fungus-treated straw was not affected by the pasteurization methods. Aflatoxin B1 was within the safe level (<5 µg kg-1 ) in all pasteurized, fungus treated straws. CONCLUSION: Steam and lime were promising field-applicable pasteurization techniques to produce nutritionally improved fungus-treated wheat straw to feed ruminants. Lime pasteurization was more economical and did not require expensive energy inputs. © 2023 Society of Chemical Industry.

Improving the nutritional value and digestibility of wheat straw, rice straw, and corn cob through solid state fermentation using different Pleurotus species.

BACKGROUND: The growing food-feed-fuel competition, declining availability of traditional feeds, higher prices, and the urgent need to provide long-term sustainability for animal production have all triggered global research into the optimum extraction of energy and nutrients from lignin-rich plant biomass. Recent studies have shown that the Pleurotus species of white rot fungus can selectively degrade lignin in lignin-rich plant biomass; however, its effectiveness in selectively degrading lignin depends on the type of substrate and species of fungus. This study was therefore designed to treat wheat straw, rice straw, and corn cob, with Pleurotus eryngii, P. ostreatus, and P. florida for 30 days under solid-state fermentation, to identify a promising fungus-substrate combination for the selective degradation of lignin and optimal improvement in the nutritional value and digestibility of each substrate. RESULTS: The type of fungus strongly influenced (P < 0.01) selectivity in lignin degradation, and the level of improvement in crude protein (CP), in vitro dry matter digestibility (IVDMD), and in vitro gas production (IVGP), in wheat straw, rice straw, and corn cob. Fungus-substrate interaction data revealed that P. ostreatus caused maximum (P < 0.05) degradation of lignin, and greater (P < 0.05) improvement in CP, IVDMD, and IVGP in wheat straw and rice straw. The lowest (P < 0.05) degradation of lignin and improvement in CP, IVDMD, and IVGP was caused by P. eryngii in corn cob. Among the fungi, the maximum (P < 0.05) degradation of lignin, and greater (P < 0.05) improvement in CP, IVDMD, and IVGP were caused by P. florida as compared with those of P. ostreatus and P. eryngii. CONCLUSION: The results highlight significant influence of fungus-substrate combination for selective lignin degradability and the consequent improvement in the nutritional value of the substrates. Maximum selective lignin degradability and improvement in nutritional value and digestibility was caused by P. ostreatus in wheat straw and in rice straw, and by P. florida in corn cob. © 2021 Society of Chemical Industry.

Genome-wide identification and analysis of soybean acyl-ACP thioesterase gene family reveals the role of GmFAT to improve fatty acid composition in soybean seed.

KEY MESSAGE: Soybean acyl-ACP thioesterase gene family have been characterized; GmFATA1A mutants were discovered to confer high oleic acid, while GmFATB mutants presented low palmitic and high oleic acid seed content. Soybean oil stability and quality are primarily determined by the relative proportions of saturated versus unsaturated fatty acids. Commodity soybean typically contains 11% palmitic acid, as the primary saturated fatty acids. Reducing palmitic acid content is the principal approach to minimize the levels of saturated fatty acids in soybean. Though high palmitic acid enhances oxidative stability of soybean oil, it is negatively correlated with oil and oleic acid content and can cause coronary heart diseases for humans. For plants, acyl-acyl carrier protein (ACP) thioesterases (TEs) are a group of enzymes to hydrolyze acyl group and release free fatty acid from plastid. Among them, GmFATB1A has become the main target to genetically reduce the palmitic acid content in soybean. However, the role of members in soybean acyl-ACP thioesterase gene family is largely unknown. In this study, we characterized two classes of TEs, GmFATA, and GmFATB in soybean. We also denominated two GmFATA members and discovered six additional members that belong to GmFATB gene family through phylogenetic, syntenic, and in silico analysis. Using TILLING-by-Sequencing+, we identified an allelic series of mutations in five soybean acyl-ACP thioesterase genes, including GmFATA1A, GmFATB1A, GmFATB1B, GmFATB2A, and GmFATB2B. Additionally, we discovered mutations at GmFATA1A to confer high oleic acid (up to 34.5%) content, while mutations at GmFATB presented low palmitic acid (as low as 5.6%) and high oleic acid (up to 36.5%) phenotypes. The obtained soybean mutants with altered fatty acid content can be used in soybean breeding program for improving soybean oil composition traits.

TILLING-by-Sequencing+ Reveals the Role of Novel Fatty Acid Desaturases (GmFAD2-2s) in Increasing Soybean Seed Oleic Acid Content.

Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; GmFAD2-1A and GmFAD2-1B. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from GmFAD2-1 alleles were used. Besides the soybean fatty acid desaturase (GmFAD2-1) subfamily, the GmFAD2-2 subfamily is composed of five members, including GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E. Segmental duplication of GmFAD2-1A/GmFAD2-1B, GmFAD2-2A/GmFAD2-2C, GmFAD2-2A/GmFAD2-2D, and GmFAD2-2D/GmFAD2-2C have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E members in controlling the seed oleic acid content. Most importantly, unlike GmFAD2-1 members, subcellular localization revealed that members of the GmFAD2-2 subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.

The effect of different ultraviolet-C light doses on microbial reduction and the components of camel milk.

As a result of increasing interest in non-thermal technologies as a possible alternative or complementary to milk pasteurization processing, the objectives of this study were to determine the effects of different ultraviolet-C light doses on the viability of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium and chemical changes to camel milk components. Pasteurized and inoculated camel milk samples were ultraviolet-C treated in a continuous flow system. The viability of E. coli O157:H7 and S. Typhimurium was evaluated with both in vivo imaging system and traditional plate count agar method. Samples subjected to the 4.15, 8.30, and 12.45 mJ/cm2 of ultraviolet-C treatment resulted in 1.9, 3.3, and 3.9-log reductions in E. coli O157:H7 and 0.9, 3, and 3.9-log reductions in S. Typhimurium, respectively. The measurement of secondary lipid peroxidation products (or ThioBarbituric Acid Reactive Substance values) showed no significant (P > 0.05) differences between the raw and ultraviolet-C treated milk samples. Additionally, no changes (P > 0.05) in the protein profiles of αs1-casein, α-lactalbumin, and lactoferrin were observed between both samples. Compared to the untreated raw milk, c9t11 conjugated linoleic acid decreased (P < 0.01) while t10c12 conjugated linoleic acid increased (P < 0.01) in the ultraviolet-C treated milk. Furthermore, three new volatile compounds were identified in the ultraviolet-C treated milk compared to the control. In conclusion, milk treated with the ultraviolet-C light at a dose of 12.45 mJ/cm2 did not meet the Food and Drug Administration (FDA) requirements for the 5-log pathogen reduction. The ultraviolet-C treatment, on the other hand, had minimal effects on camel milk components.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Effect of alkaline and sonication pretreatments on the rumen degradability of date palm seeds.

The main objective of this research was to evaluate the effects of chemical treatment and sonication (ultrasound) processing on the fiber composition and rumen degradability of date palm seeds (DPS). In the first trial, the effects of incubation or sonication in 4% sodium hydroxide (NaOH) on DPS fiber content and ruminal degradability were evaluated. Relative to untreated seeds, the ruminal degradability of DPS neutral detergent fiber (NDF) and organic matter (OM) increased (P < 0.05) for the treated seeds and were highest (P < 0.05) for the sonicated seeds. Relative to untreated seeds, the hemicellulose and lignin content were lower (P < 0.05) for the sonicated seeds, while the cellulose content was higher (P < 0.05) for the incubated seeds. In the second trial, the effects of subjecting DPS to different sonication times (5, 10, 20, and 30 min) were evaluated. The degradability of seeds' NDF and OM were greater (P < 0.05) for the sonicated than unsonicated seeds. The highest NDF degradability was seen after 30 min sonication, whereas the OM degradability was not affected by sonication time (P > 0.05). In the third trial, the effects of subjecting DPS to sonication in different NaOH solutions (1%, 2%, 4% NaOH) were evaluated. Relative to untreated seeds, the rumen degradability of seeds' NDF and OM increased with all NaOH concentrations but was highest (P < 0.05) with the 4% NaOH. In conclusion, our results showed that treating DPS with 4% NaOH increased the seeds' ruminal degradability, and subjecting DPS to sonication further improved their degradability in the rumen.

Effects of Natural Antioxidants on The Stability of Omega-3 Fatty Acids in Dog Food.

INTRODUCTION: The efficiency of five natural antioxidants (curcumin, cranberry, pomegranate, grape seed extract (GSE), and açai berry) in reducing lipid oxidation in dog food was compared to that of the synthetic antioxidant butylated hydroxyanisole (BHA). MATERIAL AND METHODS: In two different experiments content parameters were measured after 12 days of storage at 55°C. In experiment one, the natural antioxidants were added at 0.2% and BHA at 0.02% of the food (DM basis), and samples were analysed for thiobarbituric acid-reactive substances (TBARS). In experiment two, the effects of GSE and curcumin at two admixture proportions (0.1% and 0.2% of food DM) on omega-3 fatty acid (FA) content were evaluated. RESULTS: TBARS values were lower than the control (P < 0.01) for curcumin, cranberry, pomegranate, and GSE but not for the açai berry (P > 0.05). By day 12, although there were no significant differences (P > 0.05) between the two curcumin treatments, they preserved higher concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P < 0.05) than the BHA and control treatments. The addition of GSE or BHA to dog food held (P < 0.05) the concentrations of EPA higher than the control. The concentrations of EPA and DHA for the 0.2% GSE treatment were greater (P < 0.05) than the 0.1% GSE treatment. Grape seed extract at 0.2% lost less (P < 0.05) EPA concentration than BHA. CONCLUSION: The present results showed that, except for açai berry, the tested natural antioxidants could be used as a substitute for BHA in dog food.

Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number.

Soybean seed oil typically contains 18-20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in FAD2-1 genes have been reported to increase seed oleic acid content. A subset of 1,037 mutant families from a mutagenized soybean cultivar (cv.) Forrest population was screened using reverse genetics (TILLING) to identify mutations within FAD2 genes. Although no fad2 mutants were identified using gel-based TILLING, four fad2-1A and one fad2-1B mutants were identified to have high seed oleic acid content using forward genetic screening and subsequent target sequencing. TILLING has been successfully used as a non-transgenic reverse genetic approach to identify mutations in genes controlling important agronomic traits. However, this technique presents limitations in traits such as oil composition due to gene copy number and similarities within the soybean genome. In soybean, FAD2 are present as two copies, FAD2-1 and FAD2-2. Two FAD2-1 members: FAD2-1A and FAD2-1B; and three FAD2-2 members: FAD2-2A, FAD2-2B, and FAD2-2C have been reported. Syntenic, phylogenetic, and in silico analysis revealed two additional members constituting the FAD2 gene family: GmFAD2-2D and GmFAD2-2E, located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other FAD2-2 members localized on chromosomes 19 (GmFAD2-2A and GmFAD2-2B) and 03 (GmFAD2-2C). This work discusses alternative solutions to the limitations of gel-based TILLING in functional genomics due to high copy number and multiple paralogs of the FAD2 gene family in soybean.

The effect of lipid supplements on ruminal bacteria in continuous culture fermenters varies with the fatty acid composition.

A single flow continuous culture fermenter system was used in this study to investigate the influence of dietary lipid supplements varying in their fatty acid content on the DNA concentration of selected rumen bacteria. Four continuous culture fermenters were used in a 4 × 4 Latin square design with four periods of 10 d each. Treatment diets were fed at 45 g/d (DM basis) in three equal portions during the day. The diets were: 1) control (CON), 2) control with animal fat source (SAT), 3) control with soybean oil (SBO), and 4) control with fish oil (FO). Lipid supplements were added at 3% of diet DM. The concentrations of total volatile fatty acids and acetate were not affected (P>0.05) by lipid supplements. Concentrations of propionate, iso-butyrate, valerate and iso-valerate were highest (P<0.05) with the FO diet compared with the other treatment diets. The concentration of til C18:l (vaccenic acid, VA) in effluents increased (P<0.05) with SBO and FO diets and was highest with the SBO diet. The concentrations of C18:0 in effluents were lowest (P<0.05) for the FO diet compared with the other treatment diets. Concentrations of DNA for Anaerovibrio lipolytica, and Butyrivibrio proteoclasticus in fermenters were similar (P>0.05) for all diets. The DNA concentrations of Butyrivibrio fibrisolvens and Ruminococcus albus in fermenters were lowest (P<0.05) with the FO diet but were similar (P>0.05) among the other treatment diets. Selenomonas ruminantium DNA concentration in fermenters was highest (P<0.05) with the FO diet. In conclusion, SBO had no effect on bacterial DNA concentrations tested in this study and the VA accumulation in the rumen observed on the FO diet may be due in part to FO influence on B. fibrisolvens, R. albus, and S. ruminantium.

Effect of feeding calcium salts on performance of nursing Awassi ewes.

Twenty nursing Awassi ewes (BW = 50 ± 2.35 kg, age = 4.5 ± 1.2 years) with their lambs were used to evaluate the effects of feeding calcium salts in lactation diets on performance and pre-weaning growth of their lambs. Treatments were 0% calcium salts (CON) or 5% calcium salts (FAT). At the end of the study, a digestibility experiment was performed. Milk yield was greater (P < 0.05) for ewes fed the FAT diet than the CON diet. Milk composition was similar (P > 0.05) between diets. However, milk energy value (kcal/day) tended to be greater (P = 0.07) for the FAT diet than the CON diet. Concentrations of milk C18:1c9 and C20:0 were greater (P < 0.05) in ewes fed the FAT diet than the CON diet. However, concentration of trans-10, cis-12 CLA was lower (P = 0.05) in the FAT diet than in the CON diet. No differences in feed intake and body weight change were detected between diets. Digestibility of dry matter, organic matter, crude protein, ether extract, neutral detergent fiber, and acid detergent fiber were similar (P > 0.05) for diets. For lambs, weaning weight was not affected by treatments. However, average daily gain and total gain were greater (P = 0.053) for the FAT diet than the CON diet. Results suggest that supplementing lactating ewes with calcium salts at the beginning of lactation phase improves daily milk yield of ewes and pre-weaning growth of their lambs with no major negative impact on feed intake and digestibility.

Enhancement of alpha- and beta-galactosidase activity in Lactobacillus reuteri by different metal ions.

The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose. alpha-Galactosidase can digest oligosaccharides like raffinose, while beta-galactosidases can hydrolyze lactose. Therefore, selection of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective of this study was to investigate the enhancement of alpha- and beta-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na(+), K(+), Fe(2+), and Mg(2+) and 1 mM of Mn(2+) were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest alpha- and beta-galactosidase activity when grown in the medium with added Mn(2+) ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn(2+) ions lead to 27, 18% enhancement of alpha- and beta-galactosidase activity over the control group, and therefore, it could be added to the growth culture of CF2-7F to produce enhanced levels of alpha- and beta-galactosidase activity. The addition of Fe(2+) led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that 0.0274% Mn(2+) can be used to promote the production for alpha- and beta-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of alpha- and beta-galactosidase activity and have a good potential to be used in the food industry.

The production of 10-hydroxystearic and 10-ketostearic acids is an alternative route of oleic acid transformation by the ruminal microbiota in cattle.

The formation of hydroxystearic acid (HSA) and ketostearic acid (KSA) from oleic acid transformation has been documented in a variety of microbial species, including several isolated from the rumen of domesticated ruminant species. However, their ruminal production rates have not been established as influenced by fatty acid source. Dosing continuous cultures of mixed ruminal microorganisms with 1-(13C)-oleic acid increased the 13C enrichment of both HSA and KSA at 24 h postdosing, and showed that the majority (96 and 85%, respectively) of the HSA and KSA present in the 24-h samples originated from oleic acid. Several experiments using batch cultures of ruminal microorganisms showed that production of HSA and KSA was directly related to oleic acid input but was not affected by elaidic acid input, and that HSA was further metabolized to KSA but not to other fatty acids. When continuous cultures of ruminal microorganisms were supplemented with soybean oil or canola oil, production of 10-HSA + 10-KSA was related to oleic acid input but not to linoleic acid input. Daily production of 10-HSA + 10-KSA across treatments was 14.4 micromol/100 micromol oleic acid input into the cultures or 31.1 micromol/100 micromol oleic acid net loss. The results of this study quantify the formation of 10-HSA and 10-KSA from oleic acid transformation by ruminal microorganisms, and show that their accumulation in ruminal contents is directly related to the extent of oleic acid input and biotransformation by the rumen microbiota.