高效棉酚降解枯草芽孢杆菌M-4菌株的常压室温等离子体诱变及发酵工艺优化

【背景】棉粕中游离棉酚的存在制约了棉粕作为饲料蛋白源的利用,棉酚的微生物降解问题成为研究热点。本实验室前期发现枯草芽孢杆菌(Bacillus subtilis) M-4菌株具有较强的降解棉酚能力,而且已经应用于棉粕脱毒工业。【目的】进一步提高枯草芽孢杆菌M-4菌株降解棉粕中棉酚的能力,扩大棉粕在养殖业的应用领域。【方法】采用常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术对菌株M-4进行诱变,以液体培养条件下的棉酚降解率为初筛指标,筛选获得正向突变株。以棉粕固体发酵条件下的棉酚降解率为复筛指标,测定初筛获得的正向突变株实际降解棉粕中游离棉酚的能力。以棉酚残存量和棉酚降解率为检测指标,采用单因素试验优化固体条件下棉粕发酵条件,获得突变株发酵棉粕的最适工艺参数。【结果】初筛获得正向突变株19株。复筛得到一株高效的突变株MY-4-17,在棉粕固体发酵条件下其棉酚降解率高达97.15%,比菌株M-4的棉酚降解率提高了2.55%。经过5次传代培养,突变株MY-4-17的遗传稳定性良好。突变株MY-4-17发酵棉粕的最适工艺参数为...     

In situ alkaline transesterification of cottonseed oil for production of biodiesel and nontoxic cottonseed meal

The production of fatty acid methyl ester (FAME) by direct in situ alkaline-catalyzed transesterification of the triglycerides (TG) in cottonseeds was examined. The experimental results showed that the amount of cottonseed oil dissolved in methanol was approximately 99% of the total oil and the conversion of this oil could achieve 98% under the following conditions: less than 2% moisture content in cottonseed flours, 0.3-0.335mm particle size, 0.1mol/L NaOH concentration in methanol, 135:1 methanol/oil mole ratio, 40 degrees C reaction temperature and 3h reaction time. Further, the effects of co-solvent petroleum ether and methanol recycling on the cottonseed oil extraction and conversion were also investigated. The use of alkaline methanol as extraction and reaction solvent, which would be useful for extraction oil and gossypol, would reduce the gossypol content in the cottonseed meal. The free and total gossypol contents in the cottonseed meal obtained from in situ alkaline transesterification were far below the FAO standard. And the nontoxic cottonseed meal could be used as animal protein feed resources.     

Effects of diets containing gossypol on spermatogenic tissues of young bulls

Eighteen yearling beef bulls were used in a study to determine the effects of diets containing gossypol on spermatogenic tissues, Sertoli cells and Leydig cells. Bulls were randomly assigned to one of three (n = 6) isonitrogenous diet groups formulated from alfalfa hay and corn (gossypol-free control), control plus whole cottonseed and cottonseed hulls, or control plus cottonseed meal and cottonseed hulls as sources of gossypol. Testicular tissues collected were examined histologically, and tissues from bulls fed gossypol-free diets were compared with those fed diets containing cottonseed products. Following a 2-mo period (P(1)) when bulls were fed diets containing gossypol, one-half of the bulls were placed on a gossypol-free diet for an additional 2 mo (P(2)) to determine if gossypol effects were reversible. At the end of P(1), bulls fed whole cottonseed and cottonseed meal had larger (P<0.01) lumens in their seminiferous tubules, decreased (P<0.001) wall thickness in their seminiferous tubules, and a reduced (P<0.0001) number of cell layers in their seminiferous tubles, when compared with bulls fed a gossypol-free diet. These histological changes indicate detrimental effects to the spermatogenic tissues and associated cells. Following the initial two months (P(1)) of receiving feed containing gossypol, herd mates that were fed a gossypol-free diet for 2 mo (P(2)) showed improvement in histological characteristics, indicating that gossypol-induced effects were partially reversible.     

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.     

高效降解棉酚菌株的分离鉴定及诱变选育

作为一种蛋白资源, 棉籽粕因其含有毒素——游离棉酚限制了其在饲料工业中的应用。为获得能高效降解棉籽粕中棉酚的菌株, 以醋酸棉酚为唯一碳源培养基从16份样品中筛选出一株高效降解棉酚菌株(Y-2), 经生理生化及18S rDNA鉴定为Pichia guilliermondii, 此菌种为非致病酵母, 且首次报道用于棉酚的降解, 在工业生产中具有潜在的应用前景。通过紫外诱变获得一株棉酚降解率更高的突变株YUV-51。通过对突变株YUV-51发酵温度、时间及接种量的初步优化, 获得其固态发酵优化条件: 30 °C培养48 h, 接种量0.025 g湿菌体/g棉籽粕, 初始水分含量50%。为避免游离棉酚在前处理中大量降解棉籽粕, 不进行湿热处理, 经接种发酵后脱毒率可达到58%。这使微生物脱毒在实际生产中应用成为可能。     

Effects of diets containing gossypol on reproductive capacity of rainbow trout (Oncorhynchus mykiss)

We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1-5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 +/- 8 g) were fed the diets over a period of 131 days during which a general 2-fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 +/- 0.3 g/dl) in comparison to treatments 1-3 (10.3-10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2-9.8 x 10(9)/ml), motility (78-89%), and standardized (300 x 10(5) sperm/egg) fertilizing ability (18.9-22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 +/- 0.2, 11.7 +/- 4.1, 21.7 +/- 1.4, and 29.9 +/- 3.9 microg/ml, for treatments 2-5, respectively). The major portion of gossypol in blood plasma was protein-bound (81-93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02-0.18 microg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days.     

可改良棉粕蛋白的菌株选育及其发酵

棉粕蛋白质含量高达38％~50％,然而作为饲料其蛋白质品质较差,饲料利用率较低。为提高其蛋白质利用率,从自然界筛选出了能够高效降解棉粕大分子蛋白质的优良菌株4株,经生化与分子生物学试验鉴定为Bacillus subtilis(2株,编号为H1和H7),Bacillus cereus (1株,编号为H9)和Aspergillus niger (1株,编号为P1)。通过单菌及组合发酵实验比较发现:最适宜菌种组合为B. subtilis(H1)和A. niger (P1)。两菌株组合发酵后,棉粕TCA-NSI(三氯乙酸氮溶指数)提高了25.34%,小分子多肽含量提高了11%,游离必需氨基酸提高了24%,蛋白质的体外消化率由44.56%提高到了78.61%,明显高于其他菌种组合,研究结果表明其组合发酵可有效改良棉粕蛋白品质,显著提高棉粕蛋白的饲用价值。     

Value-added subcritical water hydrolysate from rice bran and soybean meal

New value-added product was derived from agricultural by-products: rice bran and soybean meal by means of subcritical water (SW) hydrolysis. The effect of temperature (200-220 degrees C), reaction time (10-30 min), raw material-to-water weight ratio (1:5 and 2:5), was determined on the yields of protein, total amino acids, and reducing sugars in the soluble products. The suitable hydrolysis time was 30 min and the proper weight ratio of the raw material-to-water was 1:5. The reaction temperature suitable for the production of protein and amino acids was 220 degrees C for raw and deoiled rice bran, 210 degrees C for raw soybean meal, and 200 degrees C for deoiled soybean meal. The products were also found to have antioxidant activity as tested by ABTS(.)(+) scavenging assay. In addition, sensory evaluation of milk added with the hydrolysis product of deoiled rice bran indicated the potential use of the product as a nutritious drink.     

复合微生物固态发酵对棉籽饼粕脱毒及营养的影响

研究了黑曲霉与酿酒酵母混合固态发酵对棉籽饼粕的脱毒效果及提高棉籽饼粕营养价值的作用。结果表明,混合发酵优化后的条件为:发酵温度30℃,接种量15%,初始水分含量0.96g水/g干基,初始pH值为6,发酵时间60 h;发酵后棉籽饼粕中游离棉酚脱毒率为95.51%,真蛋白质含量从32.99%提高到 42.40%,必需氨基酸中赖氨酸、蛋氨酸和苏氨酸分别较发酵前相比增加了31.03%、25.00%和44.23%,氮溶解指数(NSI)增加了1.49倍,从而改善了棉粕饼粕的品质。     

Effects of diets containing gossypol on ovarian histology, function and fertility in prepubertal beef heifers

Forty-five crossbred beef heifers (weight = 268.3 +/- 5.7 kg) were used to determine the effects of dietary gossypol on ovarian morphology, erythrocyte fragility and fertility. Heifers were randomly assigned to 1 of 3 isonitrogenous dietary treatments. The diet consisted of rice mill feed and milo supplemented with soybean meal (n = 13; control), cottonseed meal (n = 16; low supplementation) which supplied 6.1 g free gossypol animal(-1) day(-1), or whole cottonseed (n = 16; high supplementation) which supplied 13.7 g free gossypol animal(-1) day(-1). The heifers were group-fed each diet for 64 days and were maintained on similar but separate fescue pastures overseeded with wheat. After 64 days, 4 heifers from each diet were confined and fed their respective diets. On Day 10 following estrus, each animal was unilaterally ovariectomized, and the ovary containing the corpus luteum was removed. The remaining ovary was removed 6 to 12 hours after detection of estrus in the next cycle. Erythrocyte fragility increased (P < 0.02) in heifers receiving gossypol compared with that of the controls. Cyclicity in the heifers was 81.3, 68.8 and 38.4% for high, low and control diets, respectively, at the end of the 64-day treatment period. First service conception rate, as determined by palpation per rectum, was similar among treatments (58.3, 33.3, 33.3% for high, low and control groups, respectively). Weight gain increased (P < 0.03) in control heifers compared with that of heifers receiving gossypol. Gross ovarian morphology and histology were similar for all heifers. Although gossypol produced mild toxicosis in heifers, no adverse reproductive effects could be detected from gossypol intake.     

RNAi‐mediated Ultra‐low gossypol cottonseed trait: performance of transgenic lines under field conditions

Cottonseed remains a low‐value by‐product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra‐low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ‐cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild‐type levels of gossypol and related terpenoids. Although it was a relatively small‐scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild‐type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%–8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi‐based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever‐increasing needs of humanity.     

Effects of gossypol on rainbow trout Salmo guirdneri Richardson

Rainbow trout were fed gossypol acetate or cottonseed meals containing gossypol for 14 months. Toxic effects were indicated by growth depression, reduced hematocrit, hemo-globin, and total plasma protein. Histological changes were noted, particularly in the liver and kidney.     

Characterization of gossypol-induced sperm abnormalities in bulls

To characterize sperm abnormalities induced by gossypol in cattle, young Brahman bulls (n=8) received either cottonseed meal delivering 8.2 g free gossypol/bull/d (treatment, n=4) or isonitrogenous soybean meal (control, n=4) for 11 wk. At slaughter, semen was collected from the following extragonadal sites: mediastinum/rete testis (Site 1), caput (Site 2), corpus (Site 3) and cauda epididymis (Site 4), and ductus deferens (Site 5). At least 200 fixed spermatozoa per site were examined via differential-interference-phase contrast (DIC) microscopy, with electron microscopy (EM) being employed with select samples. Sperm midpiece abnormalities were categorized as aplastic, fragile or asymmetric, with detached sperm heads being tabulated separately. Of these, aplastic defects were considered most likely to occur during spermatogenesis. Bull sperm midpiece lesions induced by gossypol were ultrastructurally similar to those observed in other, nonruminant, species. Combined midpiece abnormalities generally increased with extragonadal passage (EGP) in the treated bulls, as did fragile and asymmetric but not aplastic midpieces, or detached sperm heads. This pattern of EGP changes in bull sperm morphology following gossypol spermatoxicity suggests that structural weakness induced during spermatogenesis leads to secondary spermatozoal changes during EGP, possibly due to the imposition of motility stressors upon prior weakened structures.     

不同饲喂下棉铃虫肠道内生菌的分离鉴定及脱毒效果研究

[目的]本研究对比饲喂正常饲料和棉粕饲料下棉铃虫(Helicoverpa armigera)肠道中可培养微生物的异同,并对筛选菌株进行棉酚耐受及降解作用的研究,为棉铃虫肠道微生物在棉源饲料中的脱毒作用提供理论和实验支持.[方法]通过饲喂不同饲料、醋酸棉酚单一碳源微生物选择性培养,分离饲喂正常饲料和棉粕饲料下棉铃虫肠道可...     

The effect of gossypol in cottonseed meal on performance and on hematological and semen traits in postpubertal Brahman bulls

The effects of a cottonseed meal diet containing high levels of free gossypol on hematological traits, including erythrocyte osmotic fragility and semen characteristics, were examined during an 11-week period. Eight Brahman bulls were randomly assigned to 1 of 2 groups. The control group (n=4) was fed a mixture of soybean meal and corn. The treated group (n=4) was fed a mixture of cottonseed meal and corn. Both groups were allowed hay free choice. The treated group consumed 8.2 g of free gossypol per bull per day. The percentage of normal spermatozoa was lower (P<0.01) in the treated than in the control group from Week 5 (49+/-9.8 vs 83+/-3.2%), which was primarily influenced by changes in mid piece morphology in the treated bulls. Erythrocyte osmotic fragility was higher (P<0.001) in the treated than in the control group over the entire study period, although group values diverged more acutely from Week 7 of treatment. Sperm motility was lower (P=0.04) in treated bulls than in control bulls at Week 9 (52+/-9.8 vs 82+/-6.2%). These data suggest that concurrent discernable changes in bull erythrocyte osmotic fragility and in semen characteristics can occur following commencement of a diet containing 8.2g of free gossypol per day.     

Simultaneous inclusion of sorghum and cottonseed meal or millet in broiler diets: effects on performance and nutrient digestibility

Two experiments were conducted to investigate the use of sorghum, cottonseed meal and millet in broiler diets and their interaction when they are used simultaneously. In Experiment 1, a corn-soybean meal control diet was compared with eight experimental treatments based on low tannin sorghum (S30, S45 and S60), cottonseed meal (CM15, CM40) or both ingredients included in the same diet (S30/CM40, S45/CM25 and S60CM15). Results showed that BW gain was not affected by the inclusion of sorghum or cottonseed meal. However, feed intake tended to be affected by the cereal type with the highest values with sorghum-based diets. Feed conversion ratio increased (P<0.001) with sorghum-based diets compared with the control diet, whereas a combination of cottonseed meal and sorghum in the same diet did not affect the feed conversion ratio. Significant differences (P<0.001) were observed in apparent ileal digestibility (%) of protein and energy with the cottonseed meal and sorghum/cottonseed meal-based diets having lower protein and energy digestibility compared with corn-based diets. In Experiment 2, a control diet was compared with six diets in which corn was substituted at 60%, 80% or 100% by either sorghum or millet and other three diets with simultaneous inclusion of these two ingredients (S30/M30, S40/M40, S50/M50). Single or combined inclusion of sorghum and millet resulted in similar feed intake and growth performance as the control diet. Apparent ileal digestibility of protein and energy was higher with millet-based diets (P<0.001). Total tract digestibility of protein in sorghum and millet-based diets tended to decrease linearly with the increasing level of substitution. Sorghum-based diets resulted in lower total tract digestibility of fat compared with millet and sorghum/millet-based diets (P<0.001). Higher total tract digestibility of starch were obtained with the control diet and millet-based diets compared with the sorghum-based treatments. Results of the two experiments suggest that broiler growth performance was not affected by the dietary level of sorghum, millet or cottonseed meal. Nutrient digestion can, however, be affected by these feed ingredients.     

Effects of dietary gossypol on aspects of semen quality, sperm morphology and sperm production in young Brahman bulls

Eight young reproductively normal Brahman bulls (average age and bodyweight 20 months and 500 kg, respectively) received either cottonseed meal delivering 8.2 g free gossypol/bull/d (treatment group, n=4) or soybean meal (control group, n=4) for 12 wk. After adjustment (1 wk), weekly procedures (11 wk) included blood collection, scrotal circumference measurement and electroejaculation. Semen assessments included sperm motility, percentage of live spermatozoa, general sperm morphology (using brightfield microscopy), and midpiece morphology (using DIC microscopy). After sacrifice (Week 12), sperm production rates (daily and per gram testicular parenchyma) were determined. Treated bulls did not differ from controls in scrotal circumference or the percentage of live spermatozoa. Sperm motility differed at Weeks 9 (P<0.05), 10 and 11 (both P=0.06). Treated bulls had fewer normal spermatozoa at Weeks 5 (P<0.05), 6 (P<0.01) and 7 thru 11 (P<0.001). Beginning from Week 3, treated bulls showed an increased proportion of sperm midpiece abnormalities (P<0.05) which stabilized at 52 to 62.5% between Weeks 5 and 11 (P<0.01 or P<0.001). Treated bulls also had lower sperm production than untreated bulls, both on a daily (P<0.01) and per gram testicular parenchyma (P<0.001) basis. A cottonseed supplement providing 8.2 g of free gossypol per bull per day had adverse effects upon both sperm morphology and spermatogenesis in young Brahman bulls, with the former being first evident within 3 to 4 weeks of feeding of cottonseed meal.     

Utilization of commercial oilseed crops

The United States is a major producer of many different types of oilseeds, but the predominant one is soybean, that remarkable legume whose meal and oil serve many animal feed, human food, and domestic industrial product needs. More than half of the soybeans and the products produced from them are exported. The 16 mill MT of soy meal processed and fed in the United States in 1981 constituted 88% of the total oilseed meal, 71% of the high-protein feeds, and 48% of total processed feeds. Of the total soy protein available, less than 5% goes into human food products such as meat extenders, simulated meats, baked goods, dairy product analogs, dietary foods, infant foods, and fermented food products. Less than 1% of soy protein in the United States is used in industrial products, mainly as a binder for pigmented paper coatings. Of the total soy oil available, about 95% is consumed in food products such as margarines, salad oils, and cooking oils. About 5% of soy oil is applied to nonfood uses such as alkyd paints, plasticizer/stabilizers for vinyl plastics, soaps, eraser factices, and many other lesser uses. Other major oilseeds produced in the United States include cottonseed, flaxseed, peanut, safflower, and sunflower. Corn oil is produced in significant quantities as a by-product of the corn starch industry. The oilseed crops having the greatest oil productivity are peanut and sunflower. However, sunflower meal has certain deficiencies for feed and food uses. If the United States is to draw upon oilseed crops as significant contributors to feed, food, industrial products, and agricultural fuel needs, greatly improved productivity will be needed either from new oilseed crops or from improved varieties of present commercial crops.     

Composition of amino acids in feed ingredients for animal diets

Dietary amino acids (AA) are crucial for animal growth, development, reproduction, lactation, and health. However, there is a scarcity of information regarding complete composition of “nutritionally nonessential AA” (NEAA; those AA which can be synthesized by animals) in diets. To provide a much-needed database, we quantified NEAA (including glutamate, glutamine, aspartate, and asparagine) in feed ingredients for comparison with “nutritionally essential AA” (EAA; those AA whose carbon skeletons cannot be formed by animals). Except for gelatin and feather meal, animal and plant ingredients contained high percentages of glutamate plus glutamine, branched-chain AA, and aspartate plus asparagine, which were 10–32, 15–25, and 8–14% of total protein, respectively. In particular, leucine and glutamine were most abundant in blood meal and casein (13% of total protein), respectively. Notably, gelatin, feather meal, fish meal, meat and bone meal, and poultry byproduct had high percentages of glycine, proline plus hydroxyproline, and arginine, which were 10–35, 9.6–35, and 7.2–7.9% of total protein, respectively. Among plant products, arginine was most abundant in peanut meal and cottonseed meal (14–16% of total protein), whereas corn and sorghum had low percentages of cysteine, lysine, methionine, and tryptophan (0.9–3% of total protein). Overall, feed ingredients of animal origin (except for gelatin) are excellent sources of NEAA and EAA for livestock, avian, and aquatic species, whereas gelatin provides highest amounts of arginine, glycine, and proline plus hydroxyproline. Because casein, corn, soybean, peanut, fish, and gelatin are consumed by children and adults, our findings also have important implications for human nutrition.     

棉籽中游离棉酚的含量测定

用紫外分光光度法测定棉籽中游离棉酚的含量。脱除棉酚的棉籽乙醚提取物经石油醚和 75 %乙醇分离和纯化后 ,用 0 .5 %对氨基苯磺酸显色来检测棉籽中游离棉酚的含量 ,结果表明 ,该方法具有简便、重现性好、回收率高达 97%以上等优点 ,可作为棉籽中游离棉酚质量控制方法。