Protein production by Schwanniomyces castellii on starchy substrates, in liquid and solid cultivation

In a preliminary screening, several yeast species were selected among which Endomycopsis fibdigera and Schwunniomyces castellii showed a high yield when inoculated in potato or potato peel media. Both these species were found to have an elevated protein percentage and a good amino acid composition. The effect of some cultural conditions on SCP (single cell protein) production by S. castellii grown on potato medium in a pilot plant 90 1 fermenter was also investigated. Optimal conditions resulted in employing a substrate containing 80 g/l of potatoes, 5.0 8/1 of cornsteep liquor, at 28°C, with a 3% inoculum.
S. castellii and E. fibdigera were also separately inoculated in solid and semi-solid substrates (potatoes, cassava flour, stale bread, maize flour). Under laboratory and semi-pilot plant conditions, carbohydrate conversion into protein was more effective on potato and cassava flour media.     

Molecular and functional analysis of a MIG1 homologue from the yeast Schwanniomyces occidentalis

A putative glucose repressor MIG1-homologue (SoMIG1) was isolated from the amylolytic yeast Schwanniomyces occidentalis. Degenerate primers were designed from the conserved zinc finger regions of Mig1 and CreA proteins from different organisms. PCR using these primers and S. occidentalis genomic DNA as template yielded a single 128 bp product. This fragment was used as a DNA probe to screen a S. occidentalis genomic library. Analysis of the positive clones led to the isolation by PCR of a DNA fragment, which contained an open reading frame (ORF) that would encode a 458 amino acid polypeptide. The DNA binding and effector domains of this putative protein showed an identity of 71% and 15%, respectively, to those of the Mig1 protein from Saccharomyces cerevisiae. The SoMIG1 gene complemented a mig1 mutant of this yeast, which suggests that in S. occidentalis SoMIG1 is a glucose repressor. The Accession No. is AJ417892.     

Effect of feeding warm-season annuals with orchardgrass on ruminal fermentation and methane output in continuous culture

A 4-unit, dual-flow continuous culture fermentor system was used to assess nutrient digestibility, volatile fatty acids (VFA) production, bacterial protein synthesis, and methane (CH₄) output of warm-season annual grasses. Treatments were randomly assigned to fermentors in a 4 × 4 Latin square design using 7 d for adaptation to treatment and 3 d for sample collection. Treatments were (1) 100% orchardgrass (Dactylis glomerata L.; ORD); (2) 50% orchardgrass + 50% Japanese millet [Echinochloa esculenta (A. Braun) H. Scholz; MIL]; (3) 50% orchardgrass + 50% brown midrib sorghum × sudangrass (Sorghum bicolor L. Moench × S. bicolor var. sudanense; SSG]; or (4) 50% orchardgrass + 25% millet + 25% sorghum × sudangrass (MIX). Fermentors were fed 60 g of dry matter (DM)/d in equal portions of herbage 4 times daily (0730, 1030, 1400, and 1900 h). To replicate a typical 12-h pasture rotation, fermentors were fed the orchardgrass at 0730 and 1030 h and the individual treatment herbage (orchardgrass, Japanese millet, sorghum × sudangrass, or 50:50 Japanese millet and sorghum × sudangrass) at 1400 and 1900 h. Gas samples for CH₄ analysis were collected 6 times daily at 0725, 0900, 1000, 1355, 1530, and 1630 h. Fermentor pH was determined at the time of feeding, and fermentor effluent samples for NH₃-N and VFA analyses were taken daily at 1030 h on d 8, 9, and 10. Samples were also analyzed for DM, organic matter (OM), crude protein, and fiber fractions to determine nutrient digestibilities. Bacterial efficiency was estimated by dividing bacterial N by truly digested OM. True DM and OM digestibilities and pH were not different among treatments. Apparent OM digestibility was greater in ORD than in MIL and SSG. The concentration of propionate was greater in ORD than in SSG and MIX, and that of butyrate was greatest in ORD and MIL. Methane output was greatest in MIL, intermediate in ORD, and lowest in SSG and MIX. Nitrogen intake did not differ across treatments, whereas bacterial N efficiency per kilogram of truly digestible OM was greatest in MIL, intermediate in SSG and MIX, and lowest in ORD. True crude protein digestibility was greater in ORD versus MIL, and ORD had lower total N, non-NH₃-N, bacterial N, and dietary N in effluent flows than MIL. Overall, we detected little difference in true nutrient digestibility; however, SSG and MIX provided the lowest acetate to propionate ratio and lower CH₄ output than MIL and ORD. Thus, improved warm-season annual pastures (i.e., brown midrib sorghum × sudangrass) could provide a reasonable alternative to orchardgrass pastures during the summer months when such perennial cool-season grass species have greatly reduced productivity.     

Rumen microbial responses to supplemental nitrate. II. Potential interactions with live yeast culture on the prokaryotic community and methanogenesis in continuous culture

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH₄ emissions. In the NO₃^? reduction process, NO₂^? can accumulate, which could directly inhibit methanogens and possibly other microbes in the rumen. Saccharomyces cerevisiae yeast was hypothesized to decrease NO₂^? through direct reduction or indirectly by stimulating the bacterium Selenomonas ruminantium, which is among the ruminal bacteria most well characterized to reduce both NO₃^? and NO₂^?. Ruminal fluid was incubated in continuous cultures fed diets without or with NaNO₃ (1.5% of diet dry matter; i.e., 1.09% NO₃^?) and without or with live yeast culture (LYC) fed at a recommended 0.010 g/d (scaled from cattle to fermentor intakes) in a 2 × 2 factorial arrangement of treatments. Treatments with LYC had increased NDF digestibility and acetate:propionate by increasing acetate molar proportion but tended to decrease total VFA production. The main effect of NO₃^? increased acetate:propionate by increasing acetate molar proportion; NO₃^? also decreased molar proportions of isobutyrate and butyrate. Both NO₃^? and LYC shifted bacterial community composition (based on relative sequence abundance of 16S rRNA genes). An interaction occurred such that NO₃^? decreased valerate molar proportion only when no LYC was added. Nitrate decreased daily CH₄ emissions by 29%. However, treatment × time interactions were present for both CH₄ and H₂ emission from the headspace; CH₄ was decreased by the main effect of NO₃^? until 6 h postfeeding, but NO₃^? and LYC decreased H₂ emission up to 4 h postfeeding. As expected, NO₃^? decreased methane emissions in continuous cultures; however, contrary to expectations, LYC did not attenuate NO₂^? accumulation.     

Effects of BioChlor and Fermenten on microbial protein synthesis in continuous culture fermenters

Meta analysis models were constructed from a data-set of 15 continuous culture fermenter trials and 118 observations on studies with either BioChlor (n = 23 observations) or Fermenten (n = 95) included at 10 and 3%, respectively, of dietary dry matter (DM) to evaluate effects of the ingredients BioChlor and Fermenten (B/F) on rumen function. Digestibility of crude protein was significantly increased by 11% with B/F treatment. This was reflected in significant increases in digestibility of DM and organic matter (OM) by 3.6 and 7.9%, respectively. Increased amounts of sugar in the diet in the presence of B/F tended to reduce digestibility of non-structural carbohydrates (NSC); however, the net effect on NSC digestion was small. There was no effect of treatment on most individual volatile fatty acids (VFA) or total VFA production. Propionate production, however, was significantly reduced in treated fermenters. The main effect of B/F as well as of starch and soluble fiber when combined with the treatment was to increase propionate production; however, the interaction between B/F treatment and sugar decreased propionate production markedly, resulting in a net decrease. The acetate-to-propionate ratio increased by 6% with B/F, largely as a result of the decrease in propionate. Production of nonammonia nitrogen was 1% less in B/F-treated fermenters, and interactions between treatment and starch, sugar, or soluble fiber were significant. Treated fermenters produced 15.7% more microbial nitrogen, in association with a significant 37% increase in rumen protein digestion. Interactions between treatment and starch, soluble fiber, or sugar influenced these results. The interaction of B/F and sugar resulted in a decrease in undegradable protein N and an increase in microbial nitrogen production. Ammonia nitrogen concentrations were increased by 24.6% in treated fermenters. Efficiency of microbial nitrogen production from DM, OM, or carbohydrate was significantly increased by B/F. Sugar content increased efficiency of microbial protein production/kg of OM digested or carbohydrate digested in the presence of treatment by >10 times the increase that was attributable to the interaction of treatment with starch. Treatment with B/F reduced moles of VFA produced/kg of microbial nitrogen produced by 16%. This effect was also substantially influenced by interactions between B/F and sugar. If the fermenter results are representative of those in vivo, milk production responses to treatment with B/F will depend on amounts of starch, soluble fiber, and, particularly, sugar in diets. Milk production responses will also depend on the quality of protein in the diet and the comparative benefit that increased flux of microbial nitrogen provides. Increased digestibility of OM should allow additional ruminant production benefits.     

Effects of substrate, passage rate, and pH in continuous culture on flows of conjugated linoleic acid and trans C18:1

A dual-flow continuous culture system consisting of 4 fermenters was used in a 4 x4 Latin square design. The objective of the research was to evaluate the effects of solid dilution rate (SDR), pH, and concentration of linoleic acid (LA) in the feed mixture on the production of conjugated linoleic acid (CLA) and trans-C18:1. The 4 treatments were 1) control = pH 6.5, 1% LA, 4%/h SDR; 2) high solid dilution rate (HSDR) = pH 6.5, 1% LA, 8%/h SDR; 3) high linoleic acid (HLA) = pH 6.5, 3% LA, 4%/h SDR; and 4) low pH (LPH) = pH 5.8, 1% LA, 4%/h SDR. Inoculum was collected 6 h after feeding from a cow fed 40% alfalfa hay and 60% grain. Liquid dilution rate was held at 0.12/h. All treatments except HLA contained 2% tallow. The LA was dissolved in buffer and continuously infused into the fermenters. The CLA flows were 16.5, 20.4, 23.2, and 25.2 mg/d for control, HSDR, HLA, and LPH, respectively. Compared with control, LPH increased flows of CLA, cis-C18:1, and C18:2, and decreased flow of C18:0. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibilities were not affected by pH. The HSDR tended to increase CLA flow compared to control, possibly because a shorter solid retention time led to incomplete biohydrogenation (BH). The NDF and ADF digestibilities and bacterial numbers were reduced by HSDR. With more LA available as a substrate for CLA, HLA resulted in a higher flow of CLA than control. The HLA resulted in the highest acid detergent fiber and fatty acid digestibilities, bacterial numbers, and BH. Increasing solids passage rate, reducing pH, and increasing dietary LA appears to increase in vitro CLA production.     

Effects of replacing dietary starch with sucrose on ruminal fermentation and nitrogen metabolism in continuous culture

A dual-effluent continuous-culture system was used to evaluate the effects of partially replacing cornstarch with sucrose in a total mixed ration on ruminal fermentation and N metabolism. The 4 treatments were 0 (control), 2.5, 5.0, and 7.5% sucrose and, respectively, 7.5 (control), 5.0, 2.5, and 0% cornstarch in a total mixed ration containing 20% corn silage and 40% alfalfa silage. Fermenters were fed 4 times a day during four 9-d periods with sampling beginning on d 6. Replacing cornstarch with sucrose did not alter ruminal pH (5.97), total volatile fatty acids (VFA) (104.4 mmol/L), or the acetate to propionate ratio (2.16); however, branched-chain volatile fatty acids were higher for the control treatment compared with the 7.5% sucrose treatment. Five hours postfeeding, sucrose treatments significantly altered molar proportions of all volatile fatty acids, and acetate-to-propionate and glucogenic-to-lipogenic ratios. Digestibility of dry matter and N were not affected by treatment, but digestibility of total non-structural carbohydrates was increased with sucrose treatments. A quadratic effect was noted for neutral detergent fiber (NDF) digestibility as sucrose replaced starch. A higher NDF digestibility (66.1 vs. 59.9%) was observed for the 7.5% sucrose treatment compared with the other 2 sucrose treatments. Levels of ammonia N were within an acceptable range to support microbial protein synthesis and did not differ among treatments (mean=9.23 mg/dL). Sucrose inclusion in the total mixed ration did not affect bacterial N synthesis. Results indicate that (at the levels tested in this study) inclusion of sucrose in the diet when rumen-degradable protein is adequate does not affect ruminal fermentation.     

Essential oil and monensin affect ruminal fermentation and the protozoal population in continuous culture

The interaction of monensin and essential oil was hypothesized to suppress protozoa and methane production while maintaining normal rumen function. The objective of this study was to determine the effects of feeding monensin (MON) and CinnaGar (CIN, a commercial blend of cinnamaldehyde and garlic oil; Provimi North America, Brookville, OH) on ruminal fermentation characteristics. Continuous culture fermentors (n = 4) were maintained in 4 experimental periods in a 4 × 4 Latin square design. Four dietary treatments were arranged in a 2 × 2 factorial: (1) control diet, 37 g/d of dry matter (40 g/d at ～92.5% dry matter) of a 50:50 forage:concentrate diet containing no additive; (2) MON at 11 g/909 kg of dry matter; (3) CIN at 0.0043% of dry matter; and (4) a combination of MON and CIN at the levels in (2) and (3). Treatment had no effects on protozoal populations, concentration of NH₃N, total N flow of effluent, production of total volatile fatty acids, or flows of conjugated linoleic acid and total C18 fatty acids. The MON decreased acetate:propionate ratio and biohydrogenation of both total C18 and 18:1 cis-9 but increased protozoal generation time, concentration of peptide, and flow of 18:1 trans-11. The MON tended to decrease protozoal counts in effluent and flow of 18:0 but tended to increase propionate production. The CIN decreased true organic matter digestibility and protozoal N flow of effluent but increased nonammonia, nonmicrobial N flow. The CIN tended to decrease protozoal counts, microbial N flow, and neutral detergent fiber digestibility but tended to increase biohydrogenation of total C18, 18:2, and 18:3. The CIN tended to increase isovalerate production. The MON and CIN tended to interact for increased methane production and bacterial N flow. A second experiment was conducted to determine the effects of MON and CIN on protozoal nitrogen and cell volume in vitro. Four treatments included (1) control (feed only), (2) feed + 0.0043% dry matter CIN, (3) feed + 2.82 μM MON, and (4) feed + CIN + MON at the same levels as in (2) and (3). With no interactions, MON addition decreased percentage of protozoa that were motile and tended to decrease cell volume at 6 h. The CIN did not affect cell count or other indicators of motility or volume at either 3 or 6 h. Under the conditions of our study, we did not detect an additive response for MON and CIN to decrease protozoal counts or methane production. A 3-dimensional method is suggested to better estimate protozoal cell volume.     

Investigation of ammonium lactate supplementation on fermentation end products and bacterial assimilation of nitrogen in dual-flow continuous culture

Supplements investigated throughout the present study are produced by fermenting lactose that is present in whey to lactate, yielding products differing in ammonium relative to lactate concentrations and in physical form (liquid or dry). Trials 1 and 2 investigated Lacto-Whey (LW; Fermented Nutrition Corp., Luxemburg, WI) and GlucoBoost (GB; Fermented Nutrition Corp.), respectively, using dual-flow continuous culture systems (n = 4), each with a 4 × 4 Latin square design. A greater proportion of nonprotein nitrogen was present in GB than in LW. In trial 1, the treatment with LW was isonitrogenously dosed against soybean meal (SBM) as a control (no LW) and factorialized with either a wheat- or corn-based concentrate (55% inclusion rate, dry matter basis). We hypothesized that LW would increase propionate production and that the combination of +LW with wheat would increase bacterial assimilation of NH₃-N into cellular N. No differences were observed for total volatile fatty acid (VFA) production per day. However, treatment × time interactions revealed that +LW increased lactate concentration at 0, 0.5, and 1 h and tended to increase molar percentage of propionate at 1 and 1.5 h postfeeding, documenting the immediate availability of lactate converted to propionate in the +LW treatments. The main effect of corn increased the proportion of bacterial N derived from NH₃-N. Trial 2 was designed to investigate GB; isonitrogenous treatments included an SBM control, crystal GB, liquid GB (LGB), and LGB with yeast culture, which were dosed twice daily. We hypothesized that GB would increase propionate production and bacterial assimilation of NH₃-N; the combination of LGB and yeast culture was expected to have a positive additive effect, yielding the greatest VFA production and bacterial NH₃-N assimilation. No differences were observed for total VFA production; however, LGB decreased molar percentage of acetate and increased propionate and butyrate molar percentages. There were no differences in non-NH₃-N flow or microbial N flow. Under the conditions of our studies, lactate in LW and GB was fermented extensively to propionate, and microbial protein synthesis in these treatments was comparable with that in SBM controls.     

Isolation and characterization of a phosphoprotein phosphatase-deficient mutant in yeast

The ppd1 mutant of yeast, Saccharomyces cerevisiae, was isolated as a suppressor of the cyr2 mutation which caused alteration of the catalytic subunit of cAMP-dependent protein kinase. Three peaks of phosphoprotein phosphatase activity (peak I, II and III) were identified by DEAE-Sephacel chromatography of crude extracts of the wild-type strain. The ppd1 mutant was deficient in peak III phosphoprotein phosphatase activity. The peak III enzyme efficiently utilized the phosphorylated forms of NAD-dependent glutamate dehydrogenase and trehalase as substrate. The ppd1 mutation did not suppress the cyr1, CYR3 or ras1 ras2 mutations. The ppd1 locus was located on chromosome II and had identical characteristics with glc1. The ppd1 mutation suppressed the G1 arrest caused by nutritional limitation, but maintained sensitivity to mating pheromone. In diploids homozygous for the ppd1 mutation, no premeiotic DNA replication and commitment to intragenic recombination occurred and no spores were formed, suggesting that the accumulation of phosphorylated proteins in the absence of one of the phosphoprotein phosphatases is required for mitosis but not for the initiation of meiosis.     

Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of α and β globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.     

Digestion during continuous culture fermentation when replacing perennial ryegrass with barley and steam-flaked corn

The objective of this study was to quantify the optimal inclusion rate of grain required to maximize nutrient digestion of a diet based on highly digestible pasture. It was hypothesized that maximum digestion would occur at a rate of grain inclusion that resulted in a culture pH of 6.0, reflecting the pH below which fiber digestion would be expected to be compromised. Four dual-flow continuous culture fermenters were used to establish the effects on digestion of replacing freeze-dried, highly digestible ryegrass with 0, 15, 30, and 45% of dry matter as 60% barley, 35% steam-flaked corn, and 5% molasses mix. The respective composite diets were fed twice daily to mimic intake patterns observed in dairy cows offered supplements during milking and offered half their daily allowance of pasture after each milking. Digesta samples were collected during the last 3 d of each of four 9-d experimental periods. Average daily culture pH decreased linearly as proportion of cereal grain in the diet increased, with average daily pH ranging from 6.29 to 5.74. Concentrations of neutral detergent fiber and total fatty acids decreased linearly with increasing proportion of cereal grain in the diet. Digestion of organic matter (OM) was maximized at an interpolated value of 24% grain inclusion and culture pH of 6.0, but the difference in the OM digestibility over the range of grain treatments from 0 to 45% was small (3 percentage units) despite pH changes over a range of 6.3 to 5.7. The relatively small change in OM digestibility was explained by reduced fiber and crude protein digestibilities being balanced by an increased digestion of nonstructural carbohydrate. Although different relationships between ruminal pH and digestibility appear to exist when cows are fed pasture alone compared with a total mixed ration, when starch supplements are included in pasture diets, the relationships associated with feeding a total mixed ration may then be more likely to apply.     

Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.     

Influence of yeast culture on ruminal microbial metabolism in continuous culture

A continuous culture study was conducted to evaluate the effect of two different yeast cultures on ruminal microbial metabolism. The treatments were a) control lactation ration, b) yeast culture 1 (YC1, Diamond-V XP) and c) yeast culture 2 (YC2, A-Max), both fed at an equivalent of 57 g/head per day. The results showed that both yeast culture products increased dry matter (DM) digestion, propionic acid production, and protein digestion compared with the control. Yeast culture 1 demonstrated an increase in molar percentage of propionic acid, a reduction in acetic acid, and a lower mean nadir (daily low) pH compared with YC2. Ruminal cultures treated with YC digested more protein and contributed less bypass N than control. Supplementing YC2 resulted in a tendency for higher microbial N/kg DM digestion than YC1. Yeast culture 1 resulted in production of rumen microbes containing less protein and more ash than YC2. These results support previous research findings that yeast culture does influence microbial metabolism, and specific yeast cultures may have different modes of action.     

Enhancement of functional characteristics of mixed lactic culture producing nisin z and exopolysaccharides during continuous prefermentation of milk with immobilized cells

Antagonistic phenomena between strains often occur in mixed cultures containing a bacteriocinogenic strain. A nisin Z producer (Lactococcus lactis ssp. lactis biovar. diacetylactis UL719) and 2 nisin-sensitive strains for acidification (Lactococcus lactis ssp. cremoris ATCC19257) and exopolysaccharide (EPS) production (Lactobacillus rhamnosus RW-9595M) were immobilized separately in gel beads and used to continuously preferment milk at different temperatures, with pH controlled at 6.0 by fresh milk addition. The process showed high volumetric productivity, with an increase from 8.0 to 12.5 L of prefermented milk per liter of reactor volume and hour as the temperature was increased from 27 to 35°C. Lactococcus lactis ssp. lactis biovar. diacetylactis UL719 counts in prefermented and fermented (22-h batch fermentation) milks were stable during 3 wk of continuous fermentation (8.1 ± 0.1 and 8.9 ± 0.2 log cfu/mL, respectively). The L. lactis ssp. cremoris population (estimated with real-time quantitative PCR) decreased rapidly during the first week of continuous culture to approximately 4.5 log cfu/mL and remained constant afterward. Lactobacillus rhamnosus counts in prefermented and fermented milks significantly increased with prefermentation time, with no temperature effect. Nisin Z reached high titers in fermented milks (from 177 to 363 IU/mL), with EPS concentration in the range from 43 to 178 mg/L. Immobilization and continuous culture led to important physiological changes, with Lb. rhamnosus becoming much more tolerant to nisin Z, and Lb. rhamnosus and L. lactis ssp. lactis biovar. diacetylactis UL719 exhibiting large increases in milk acidification capacity. Our data showed that continuous milk prefermentation with immobilized cells can stimulate the acidification activity of low-acidifying strains and produce fermented milks with improved and controlled functional properties.     

粘质沙雷氏菌的分离鉴定及产红色素培养基的优化

为确定KMR-3菌株的分类地位,对其进行了分类鉴定;为了提高其红色素产量,对发酵培养基进行了优化。以分离到的KMR-3菌株为供试材料,通过形态学、生理生化特征和16S rRNA基因序列分析对其进行了生物学分类鉴定。以LB液体培养基作为基础培养基,通过优化其不同组分（蛋白胨,酵母膏和NaCl）及含量以提高红色素产量。结果表明:KMR-3菌株归属为粘质沙雷氏菌,该菌株产红色素的最佳培养基为5g/L胰蛋白胨和1%甘油,pH5.0,温度28℃;该条件下,色素产量是基础培养基的13.4倍。该培养基蛋白用量少,促进粘质沙雷氏菌产生大量红色素,为其大规模生产提供依据。      

Evaluation of continuous lactation and increased milking frequency on milk production and mammary cell turnover in primiparous Holstein cows

We hypothesized that early-lactation increased milking frequency, in combination with bovine somatotropin (bST), would improve milk yield in continuously milked (CM) primiparous glands through greater mammary epithelial cell (MEC) function, proliferation, and reduced apoptosis (cell turnover). Primiparous cows were randomly assigned to a 2 × 2 × 2 factorial with a split-plot design to either a continuous bST (+bST, n = 4) or no bST (−bST, n = 4) treatment throughout the study. Within each animal, udder halves were randomly assigned to either a CM or a 60-d dry period (control). During late gestation, CM glands were milked twice daily until calving or until spontaneous dry-off. At calving, cows were milked either twice or 4 times daily and udder-half milk yield was recorded until 30 d postpartum. Mammary biopsies were conducted on −19 ± 13, −8 ± 6, +2, +7, and +20 d relative to calving. Postpartum milk yield was reduced in CM udder halves. Reduced milk yield in CM half udders from cows administered bST and milked 4 times daily was 35% compared with 65% in CM half udders in cows not provided bST and milked twice daily. Proliferation of MEC tended to be greater in control vs. CM tissue at 8 ± 6 d prepartum. Mammary epithelial cell proliferation was greater during the prepartum period (d −19, −8) compared with postpartum time points (d 2, 7, 20). Apoptosis of MEC was not affected by dry period length, but was elevated during the first 7 d postpartum compared with levels measured at −19, −8, and 20 d. Bovine somatotropin did not alter MEC turnover in primiparous CM or control glands. The use of increased milking frequency and bST alleviated, but did not prevent, reductions in milk yield of CM primiparous cows.     

Association of aqueous hydrogen concentration with methane production in continuous cultures modulated to vary pH and solids passage rate

The objective of this study was to evaluate the effects of altering pH and solids passage rate (k_p) on concentration of aqueous H₂ [H₂(aq)], CH₄ production, volatile fatty acids (VFA) production, and fiber digestibility in a continuous culture fermentation system. The present study was conducted as a 2 × 2 factorial treatment arrangement in a Latin square design using continuous culture fermentors (n = 4). Our continuous culture system was converted to a closed system to measure CH₄ and H₂ emission while measuring H₂(aq) concentration and VFA production for complete stoichiometric assessment of fermentation pattern. Treatments were control pH (CpH; ranging from 6.3 to 6.9) or low pH (LpH; 5.8 to 6.4) factorialized with solids k_p that was adjusted to be either low (Lk_p; 2.5%/h) or high (Hk_p; 5.0%/h); liquid dilution was maintained at 7.0%/h. Fermentors were fed once daily (40 g of dry matter; 50:50 concentrate:forage diet). Four periods lasted 10 d each, with 3 d of sample collection. The main effect of LpH increased nonammonia nitrogen flow, and both LpH and Hk_p increased nonammonia nonbacterial N flow. We observed a tendency for Hk_p to increase bacterial N flow per unit of nonstructural carbohydrates and neutral detergent fiber degraded. The main effect of LpH decreased H₂(aq) by 4.33 µM compared with CpH. The main effect of LpH decreased CH₄ production rate from 5 to 9 h postfeeding, and Hk_p decreased CH₄ production rate from 3 to 9 h postfeeding. We found no effect of LpH on daily CH₄ production or CH₄ produced per gram of neutral detergent fiber degraded, but Hk_p decreased daily CH₄ production by 33.2%. Both the main effects of LpH and Hk_p decreased acetate molar percentage compared with CpH and Lk_p, respectively. The main effect of both LpH and Hk_p increased propionate molar percentage, decreasing acetate-to-propionate ratio from 2.62 to 2.34. We noted no treatment effects on butyrate molar percentage or total VFA production. The results indicate increasing k_p and decreasing pH decreased acetate-to-propionate ratio, but only increasing k_p decreased CH₄ production; lack of differences for LpH might be a result of compensatory methanogenesis during the second half of the day postfeeding.