Effective gossypol removal from cottonseed meal through optimized solid-state fermentation by Bacillus coagulans.

BACKGROUND: Cottonseed meal (CSM) is the main by-product of the cottonseed oil extraction process with high protein content, which is an important protein source for feed industry. However, CSM contains free gossypol (FG), a toxic substance that is detrimental to animal health and greatly limits its application. Microbial fermentation is currently considered to be one of the most effective methods to reduce FG and other anti-nutritional factors in CSM. Previously, yeast and bacteria species are used for degradation of FG in CSM, but showing less detoxification efficiency. Bacillus coagulans combines the properties of both lactic acid bacteria and Bacillus, producing both lactic acid and spores, and is considered a potential probiotic. In this study, we aimed to evaluate and optimize the effect of the solid-state fermentation process using a Bacillus coagulans to gossypol removal contained cottonseed meal. RESULTS: 36 B. coagulans strains were isolated and found to have the ability to remove free gossypol. Through the evaluation of strains and optimization of fermentation conditions including fermentation temperature, ratio of material to water, inoculation amount, fermentation time and pH, we have established a solid-state fermentation process using a Bacillus coagulans strain S17 on CSM substrate with 1:1 of the material-to-water ratio, 15% (v/w) seed inoculation, 2% expanded corn flour, 1% bran, and 0.3%-0.8% metal irons at 40 °C for 52 h. After fermentation, the FG content in CSM was reduced from 923.80 to 167.90 mg/kg with 81.83% detoxification efficiency. Meanwhile, the crude protein content in CSM increased from 47.98 to 52.82%, and importantly, the spore concentration of strain S17 reached 1.68 × 1010 CFU/g dry material. CONCLUSION: The study showed that B. coagulans have the potential strong ability to degrade free gossypol through cottonseed meal fermentation. This study presents a feasible process for improving the resource utilization rate and nutritional value of CSM via solid-state fermentation through B. coagulans S17.

Enzymological, histological, and serum biomarker responses of snubnose pompano on complete replacement of fishmeal using cottonseed meal supplemented with lysine and methionine in the diet.

In a feeding experiment, cottonseed meal (CSM) was used to replace fishmeal (FM) in the diet of snubnose pompano, Trachinotus blochii, supplemented with lysine and methionine to assess the growth, nutritive profile, hematological, histological, and stress biomarker response. Experimental fishes were randomly stocked in five treatments each with triplicates. Five isonitrogenous and isolipidic diets with graded level of CSM (0, 8.7, 17.4, 26.0, and 34.7%) as replacement for FM protein (0, 25, 50, 75, and 100%) were formulated and fed to respective treatments. Comparison between various parameters among the treatments was made using orthogonal polynomial contrasts to indicate the statistical significance. Higher alkaline phosphatase, acid phosphatase, lactate dehydrogenase, malate dehydrogenase, aspartate, and alanine aminotransferase activities were observed in 0CSM group and followed by 100CSM group as higher inclusion level of CSM with higher free gossypol content did not affect the metabolic enzyme activities. The maximum muscular free gossypol accretion of 1.28 mg kg-1 (on wet basis) was recorded in 100CSM group which was very well below the critical limit set by FDA. As a conclusion, fishmeal can be completely replaced using cottonseed meal in the diet of pompano without adverse effect on growth, metabolism, and general health.

Effect of dietary cotton stalk on nitrogen and free gossypol metabolism in sheep.

OBJECTIVE: This study was to investigate the effects of dietary cotton stalk on nitrogen and free gossypol in sheep. METHODS: Treatments included 25% cotton stalk (Treat 1), 50% cotton stalk (Treat 2), and a control (no cotton stalk). Six Xinjiang daolang wethers were cannulated at the rumen and duodenum and fed one of these diets. The effects of these diets on nitrogen and free gossypol absorption and metabolism were determined. Fifteen healthy Xinjiang daolang wethers were assessed for daily gain, tissue lesions, and free gossypol accumulation. RESULTS: Dry matter intake decreased with increasing dietary cotton stalk. Total tract dry matter digestibility did not significantly differ among treatments. Dietary cotton stalk significantly decreased volatile fatty acids and increased ammonium nitrogen in the rumen. Nitrogen intake was significantly higher in Treat 2 than in the control or Treat 1. Nitrogen retention and free gossypol intake increased with dietary cotton stalk. Duodenal free gossypol flow did not increase, and free gossypol almost disappeared from the rumen. The free gossypol content of plasma and tissue was increased with dietary cotton stalk with liver free gossypol> muscle free gossypol>kidney free gossypol. Elevated dietary free gossypol decreased platelets, hemoglobin, and serum iron. Aspartate aminotransferase and γ-glutamyltransferase increased in Treat 2. With high long-term dietary cotton stalk intake, liver cells were swollen, and their nuclei dissolved. Renal cells were necrotic and the interstitia were enlarged. CONCLUSION: With short-term cotton stalk administration, only a small amount of free gossypol is retained in the body. In response to long-term or high free gossypol cotton stalk feeding, however, free gossypol accumulates in, and damages the liver and kidneys.

Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain Rumen Bacillus Subtilis.

OBJECTIVE: The aim of the study was to isolate gossypol-degrading bacteria and to assess its potential for gossypol degradation. METHODS: Rumen liquid was collected from fistulated cows grazing the experimental pasture. Approximately 1 mL of the rumen liquid was spread onto basal medium plates containing 2 g/L gossypol as the only source of carbon and was then cultured at 39°C to isolate gossypol-degrading bacteria. The isolated colonies were cultured for 6 h and then their size and shape observed by microscope and scanning electron microscope. The 16S rRNA gene of isolated colonies was sequenced and aligned using National Center for Biotechnology Information-Basic Local Alignment Search Tool. The various fermentation conditions, initial pH, incubation temperature, inoculum level and fermentationperiod were analyzed in cottonseed meal (CSM). The crude protein (CP), total gossypol (TG), and free gossypol (FG) were determined in CSM after fermentation with isolated strain at 39°C for 72 h. RESULTS: Screening results showed that a single bacterial isolate, named Rumen Bacillus Subtilis (RBS), could use gossypol as a carbon source. The bacterium was identified by 16S rDNA sequencing as being 98% homologous to the sequence of Bacillus subtilis strain GH38. The optimum fermentation conditions were found to be 72 h, 39°C, pH 6.5, moisture 50%, inoculum level 107 cell/g. In the optimum fermentation conditions, the FG and TG content in fermented CSM decreased 78.86% and 49% relative to the control. The content of CP and the essential amino acids of the fermented CSM increased respectively, compared with the control. CONCLUSION: The isolation of a gossypol-degrading bacterium from the cow rumen is of great importance for gossypol biodegradation and may be a valuable potential source for gossypol-degradation of CSM.

Effects of fermented cottonseed meal on the growth performance, gastrointestinal microflora population and small intestinal morphology in broiler chickens.

1. This experiment was conducted to evaluate the effects of replacing dietary cottonseed meal (CSM) or fermented cottonseed meal (FCSM) for soya bean meal (SBM) on growth performance, carcass characteristics, gastrointestinal microbial populations, and intestinal morphology of broiler chickens. 2. CSM was fermented with Bacillus subtilis, Aspergillus niger and A. oryzae for 7 d. A total of 300 one-d-old male Ross 308 broiler chickens were used in a 42-d experiment in which the birds were randomly allotted to one of 5 dietary treatments (containing 0%, 10% and 20% CSM or FCSM) in a completely randomised design. Birds were reared on litter floor and had free access to feed and water during the experiment. 3. Results indicated that the fermentation process significantly reduced crude fibre and free gossypol, while it increased crude protein content and lactic acid bacteria (LAB) count in CSM. 4. The use of FCSM instead of CSM significantly improved growth performance of broilers. The abdominal fat yield in treatments containing FCSM was significantly lower than in the other treatments. The increase in the population of LAB in the crop and decrease in the population of coliforms in the ileum of birds fed on diets containing FCSM were more significant than in other birds. Villi in the duodenum and jejunum of the birds fed on diets containing FCSM were significantly higher than for the other experimental groups. 5. The positive effects of diets containing FCSM on growth performance and intestinal health of broiler chickens showed that this processed source of protein can serve as an appropriate alternative for SBM in diets for broiler chickens.

Application of low-gossypol cottonseed meal in laying hens' diet.

An experiment was conducted to evaluate the application of dietary low-gossypol cottonseed meal (LCSM) in layers' diets. A total of 432 40-week-old Hy-line W36 laying hens were allocated to one of the six dietary treatments with 6 replicates of 12 birds each. The control group was fed a corn-soybean meal basal diet, and the 4 experimental diets consisted of a basal diet with 50, 98.3, 144.2, or 189 g/kg LCSM, respectively (correspondingly 25%, 50%, 75%, 100% dietary protein provided by soybean meal were replaced by LCSM). The sixth group was fed a basal diet supplemented with free gossypol (FG group). The feeding trial lasted for 12 weeks. The results showed that no significant difference was observed on egg production or feed conversion ratio (FCR, feed/egg, g/g) among all groups, but feed intake and egg weight were significantly decreased in the 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). A significant decrease was not observed in shell strength, shell thickness, and yolk color in all periods, but Haugh unit, albumen height, and egg white protein content were reduced in 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). No significant differences were found between the control and FG group. There was no obvious difference on plasma levels of total protein, blood urea nitrogen, uric acid, and activities of albumen, alanine aminotransferase, aspartate aminotransferase and the histopathology of liver, kidney, and oviduct in all groups. In conclusion, decreasing feed intake, egg weight, and egg quality were observed in the 189 g/kg LCSM group. However, the adverse effect was not relevant to the presence of FG in LCSM. Dietary LCSM supplementation of 98.3 g/kg was recommended with optimum FCR and without adverse effect on egg production, quality, and health of layers.

Effective Degradation of Free Gossypol in Defatted Cottonseed Meal by Bacterial Laccases: Performance and Toxicity Analysis.

Cottonseed meal (CSM) is the major by-product of the cottonseed oil extraction process with high protein content. However, the presence of free gossypol (FG) in CSM severely restricts its utilization in the food and animal feed industries. The development of a biological strategy for the effective removal of FG in CSM has become an urgent need. In this study, three bacterial laccases including CotA from Bacillus licheniformis, CueO from Escherichia coli, and LcLac from Loigolactobacillus coryniformis were heterologously expressed and investigated for their FG degradation ability. The results showed that CotA laccase displayed the highest FG-degrading capacity among the three laccases, achieving 100% FG degradation at 37 °C and pH 7.0 in 1 h without the addition of a redox mediator. Moreover, in vitro and in vivo studies confirmed that the hepatotoxicity of FG was effectively eliminated after oxidative degradation by CotA laccase. Furthermore, the addition of CotA laccase could achieve 87% to 98% FG degradation in defatted CSM within 2 h. In conclusion, CotA laccase can be developed as an effective biocatalyst for the detoxification of FG in CSM.

GhMYC1374 regulates the cotton defense response to cotton aphids by mediating the production of flavonoids and free gossypol.

Myelocytomatosis (MYC) transcription factors (TFs) in plants are well-known regulators of plant defense against herbivores. However, the role and mechanism of MYC TFs in cotton (Gossypium hirsutum L.) defense against cotton aphids (Aphis gossypii Glover) remain still elusive. Herein, on the basis of aphid-induced cotton transcriptome analysis, GhMYC1374, a cotton MYC2-like TF that was highly induced by cotton aphid attack, has been identified that confers cotton aphid resistance in cotton. GhMYC1374 was an intranuclear transcription factor with three domains: bHLH-MYC_N, RBR and bHLH_AtAIB_like. GhMYC1374 was induced under cotton aphid feeding, exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments. GhMYC1374 transient overexpression in cotton plants enhanced cotton aphid-resistance, while GhMYC1374 silence through VIGS (virus induced gene silencing) decreased cotton aphid-resistance. GhMYC1374 transient overexpression of in cotton plants activated the phenylpropane pathway and promoted the synthesis of flavonoids, and resistance to thus enhanced the cotton resistance against aphids. In contrast, GhMYC1374 silence inhibited the biosynthesis of flavonoids. In addition, GhMYC1374 also positively activated the expression of the biosynthetic genes of free gossypol, leading to the high content of free gossypol. Taken together, our results suggest that GhMYC1374 is involved in the cotton defense response against cotton aphids by regulating the biosynthesis of flavonoids and free gossypol.

Effect of lactic acid bacteria and yeast supplementation on anti-nutritional factors and chemical composition of fermented total mixed ration containing cottonseed meal or rapeseed meal.

OBJECTIVE: This study aimed to determine the appropriate supplementation level of lactic acid bacteria (LAB; Lactobacillus plantarum and Bacillus clausii), yeast (Saccharomyces cariocanus and Wickerhamomyces anomalus) for degrading free gossypol and glucosinolate in the fermented total mixed ration (TMR) containing cottonseed meal (CSM) or rapeseed meal (RSM), to improve the utilization efficiency of these protein sources. METHODS: For LAB, L. plantarum or B. clausii was inoculated at 1.0×108, 1.0×109, 1.0×1010, and 1.0×1011 colony-forming unit (CFU)/kg dry matter (DM), respectively. For yeast, S. cariocanus or W. anomalus was inoculated at 5×106, 5×107, 5×108, and 5×109 CFU/kg DM, respectively. The TMR had 50% moisture and was incubated at 30°C for 48 h. After fermentation, the chemical compositions, and the contents of free gossypol and glucosinolate were determined. RESULTS: The results showed that the concentration of free gossypol content was reduced (p<0.05), while that of the crude protein content was increased (p<0.05) in the TMR containing CSM inoculated by B. clausii (1×109 CFU/kg DM) or S. cariocanus (5×109 CFU/kg DM). Similarly, the content of glucosinolate was lowered (p<0.05) and the crude protein content was increased (p<0.05) in TMR containing RSM inoculated with B. clausii (1×1010 CFU/kg DM) or S. cariocanus (5×109 CFU/g DM). CONCLUSION: This study confirmed that inclusion of B. clausii with 1.0×109 or 1.0×1010 CFU/kg DM, or S. cariocanus (5×109 CFU/kg DM) to TMR containing CSM/RSM improved the nutritional value and decreased the contents of anti-nutritional factors.

Anaerobic solid-state fermentation with Bacillus subtilis for digesting free gossypol and improving nutritional quality in cottonseed meal.

Microbial fermentation is an effective method to degrade free-gossypol, which is a toxic substance restricting the utilization of cottonseed meal in animal husbandry. However, there are few researches on the nutritional effect and the change of bacterial community on cottonseed meal fermented with anaerobic solid-state fermentation. This study evaluated the effects of fermentation with Bacillus sp. on gossypol degradation and nutritional quality improvement in cottonseed meal (CM), as well as the changes of bacterial community structure during fermentation. The strains with high activity for digesting free gossypol were screened from high protease-producing strains preserved in the laboratory. Then the strains which had both the gossypol degradation activity and protease producing activity were selected to degrade macromolecular protein and free gossypol in CM. The unsterilized SSF medium was inoculated with 109 CFU/kg Bacillus culture and fermented at room temperature for 14 days. Each group had three parallels. And the effects of anaerobic solid-state fermentation on unsterilized CM was evaluated. Results showed that for the seven strains with high activity for digesting free gossypol and producing protease that were screened, free gossypol content in fermented cottonseed meal (FCM) decreased and acid-soluble protein (ASP) contents increased. Among them, strain M-15 had the best fermentation effect, with the free gossypol degradation rate of 93.46% and acid soluble protein content of 13.26%. M-15 was identified as Bacillus subtilis. During fermentation with M-15, the bacterial diversity in CM was reduced, but not significant and the community structure was simpler significantly. The strain M-15 selected in this experiment reduced the free gossypol content and improved the nutritional quality of CM through anaerobic solid-state fermentation, which can be used for industrial large-scale production.

Effects of Dietary Cottonseed Oil and Cottonseed Meal Supplementation on Liver Lipid Content, Fatty Acid Profile and Hepatic Function in Laying Hens.

Antinutrients, such as cyclopropene fatty acids (CPFAs) and free gossypol (FG), present together in cottonseed have caused numerous adverse effects on liver health and egg quality of laying hens, which are both likely to be related to a disturbance in lipid metabolism. This experiment employed a 3 × 3 factorial arrangement using corn-soybean-meal-based diets supplemented with different levels of cottonseed oil (0%, 2%, or 4% CSO) containing CPFAs and cottonseed meal (0%, 6%, or 12% CSM) containing FG to elucidate the effects of them or their interaction on fatty acid profile, lipid content, and liver health of laying hens. An overall increase in fatty acid saturation and an overall significant decrease (p < 0.05) in monounsaturated fatty acids (MUFAs) were shown in the livers of hens fed diets with either 2% or 4% CSO. Meanwhile, the concentration of liver cholesterol, serum cholesterol, and serum LDL-c of hens fed a diet supplemented with a high level of CSO (4%) were noticeably increased (p < 0.05). Even though the supplementation of 4% CSO in diets aroused beneficial influences on liver function, a high level of CSO inclusion in laying hens' diets is not recommended due to its hypercholesterolemia effect. In conclusion, supplementation of CSO, which contains 0.20% CPFAs, was the primary cause of alteration in fatty acid composition and cholesterol content in hens, while no interaction between CSM and CSO nor CSM effect was found for lipid profile and liver health in laying hen.

Presence of free gossypol in whole cottonseed.

The European Commission asked EFSA to assess information provided by the Spanish Ministry of Agriculture, Food and Environment, on the toxicity of free gossypol in relation to the use of whole cotton seed in feed for ruminants, in particular dairy cows, and, if necessary, to update the previous opinion of the EFSA Panel on Contaminants in the Food Chain (CONTAM) on gossypol as an undesirable substance in animal feed. Gossypol is a polyphenolic compound that exists in a racemic mixture of (+)-gossypol and (-)-gossypol isomers. It occurs in free or (protein-) bound forms in cottonseeds. The most commonly used cottonseeds in feed are from Upland and Pima varieties. The Pima variety is considered more toxic due to a higher content of the (-)-gossypol isomer. Upland whole cottonseeds (WCS) are fed with no further processing (after delinting); Pima varieties normally undergo further processing (grinding or cracking). It is claimed that WCS have a greater retention time in the rumen, which results in an increased detoxifying activity, compared to a shorter ruminal retention time, in the case of cracked cottonseed or cottonseed meal products. Increased erythrocyte fragility has been observed in cows given WCS Upland varieties at similar exposure levels as those resulting from an inclusion rate of 10% of WCS containing gossypol at 7,000 mg/kg in feed - the maximum permitted level of gossypol in WCS suggested by the Spanish Delegation. The information from the Spanish delegation does not differentiate between varieties in their suggestion for an increase in the maximum permitted content of free gossypol for WCS. As both Upland and Pima varieties are grown in the EU and are used for animal feed, both varieties of WCS should be considered. The CONTAM Panel considered it not necessary to update the previous opinion.