This study aimed to investigate the effect of stocking density on the behavior, productivity, and metabolism of periparturient Holstein cows as well as calf performance.A total of 48 periparturient cows were randomly assigned into three groups at 28 days (±3 days) before their expected calving date. The stocking densities of the groups, relative to the standard cubicle and feed bunk number, were i) 80% (13 cows), ii) 100% (16 cows), and iii) 120% (19 cows). Lying and rumination behavior was recorded using electronic data loggers and HR-Tags from d −21  until the calving date, d 0. Lying time was assessed to determine the diurnal total hours spent lying per day. Rumination time was averaged in 2 hours interval periods over 24 hours during the experimental period.Cows in the 80% group spent more time lying and ruminating between d −21 and d −7 and tended to ruminate more between d −14 and d 0. Calcium levels tended to be higher for cows in the 80% group, no other observable differences were found in monitored blood parameters. Moreover, 3.5% fat corrected milk and energy corrected milk yields were higher in 80% group in the first month of lactation. No other observable differences were found in the yield and composition of colostrum and milk in the first 10 months of lactation. The growth and performance of calves in the first week of life was not affected by stocking density of the dams.We concluded that lower stocking density may increase lying and ruminating behavior of prepartum Holstein cows. However, this did not translate into improved productivity and metabolism. During the transition from gestation to lactation, dairy cows experience several social stressors including regrouping and nutritional changes. If these stressors are not managed well, they could exacerbate the incidences of metabolic diseases immediately after parturition in cows . OverstoThe recommended stocking density during the prepartum period is at least one cubicle of resting space per cow . Variousin utero calf development, with almost 60% of calf growth happening in the third trimester. Although studies on stocking density are on the increase, research regarding the in utero environment of cows are scarce. A few studies on nutrition and heat stress have indicated that calves are affected by pre-partum cow management [Prepartum management may not only affect the cow and its health, but it can also have detrimental effects on nagement . It remanagement ,11. MoreAll the experimental protocols followed in this study were approved by the Institutional Animal Care and Use Committee of the China Agricultural University . The experiment was conducted between December 2017 and January 2018. In total, 48 multiparous prepartum Holstein cows  were selected and randomly assigned into three pens (20 m long and 12.8 m width) at 4 weeks (d 28±3) before parturition. All the selected cows were entering the 2nd to the 4th lactation based on their proximity to their calving dates from a dry off group of 300 cows at Zhongdi Farm in Beijing, a commercial farm with 4,500 heads of dairy cattle. Each pen was assigned to one of the three treatments and balanced for parity (2.8±0.1), previous milk yield (37.0±6.2 kg/d), and body weight . Cows were weighed on d −21  and d 1  relative to parturition. Cows had a similar body condition score (BCS) of between 3 and 5  and a locomotion score of under 2  at the time of enrollment [ad libitum intake. All cows underwent a daily clinical examination by the farm veterinarian throughout the study period. In addition, the pens were monitored via a video feed in proximity to the pens.The stocking density of the three treatments were calcu lated as the number of cows relative to the cubicle and feed bunk number, which were: i) 80% (13 cows), ii) 100% (16 cows), and iii) 120% (19 cows). One thermostatic water trough (366×56 cm) with two watering openings was available in each pen. The feed alleys and back alleys were 4.25 m and 3.70 m in width, respectively. The floor was scraped with an automatic scraper system . The cubicles were fitted with waterbeds that were resupplied with approximately 10.0 cm of rice husk once per week. The cows were provided a close up diet  feed and fresh cow diet , pre- anAfter calving, the cows were transferred to a separate pen for post-calving management and health examination. The cows received a Ca supplement  immediately after calving and received the second Bovikalc 12 h later. Cows also received an oral administration of Propylene Glycol and Ca-P-Mg  mixture during the 12 h. The calf was moved within 5 minutes after calving to a newborn calf room. Cows deemed healthy one day post-calving were moved to a free-stall pen with 240 cubicles and 260 headlocks. Calves received 4 L of colostrum within 1 h of parturition (immunoglobulin G >22 g/dL) as per the farm protocol. Calves were transferred into individual hutches bedded with straw on d 1, just before the first milk feeding, where they were monitored for 7 days after birth. Calves were reared under identical conditions. They received 3 L of pasteurized waste milk twice daily at 7:00 am and 15:00 pm. BW was recorded immediately after birth and on d 7 using a digital scale. Average daily gain was calculated by dividing the difference between initial BW and final BW by number of days.Electronic data loggers ; validated by Ledgerwood et al  were plaAfter calving, colostrum yield  was recorded. The total protein (TP) of colostrum was tested by the PAL-1 type digital Brix refractometer  and pH was analyzed by a portable pH meter . Cows were milked thrice daily. Milk yield of each cow was collected on day 15 and every 15th day of the month thereafter, post-calving and averaged as the daily milk yield. Subsequently, milk samples were collected from three time milking points and then mixed  for the analysis of milk composition  such as fat, protein, lactose, and urea nitrogen using a Combi Foss FT+ instrument . Somatic cell counts (SCC) were analyzed by a Fossomatic 5000 apparatus . Fat-corrected milk yield (FCM) was calculated using the equation: 0.432×milk yield (kg/d)+16.23×milk fat yield (kg/d); and the energy corrected milk yield (ECM) was calculated based on the following equation: 0.327×milk yield (kg/d)+12.95× milk fat yield (kg/d)+7.2×milk protein yield (kg/d).Blood samples were collected from the coccygeal vein on d −7, 0, 1, and 7 relative to the expected calving date for all cows and on d 0, 1, and 7 of age from the jugular vein for the calves. All blood samples were collected into vacuum blood collection tubes  containing no anticoagulant. The blood sampling time for both dairy cows and calves was approximately 6:00 am before the morning feeding, except for d 0 which was sampled right after calving and before colostrum feeding for cows and calves, respectively. The blood was centrifuged at 2,400×g for 15 min at 4°C. The serum was stored at −20°C in a freezer until analysis.The samples were analyzed using commercially available kits according to manufacturer’s instructions. The concentration of cortisol (COR), total cholesterol (TC), triglycerides (TG), TP, NEFA, β-hydroxybutyrate (BHBA), blood urea nitrogen (BUN), and Ca were determined in the serum. All blood samples were quantified by an automated biochemistry analyzer  except for COR that was measured by a multichannel radioimmune-counter . In calves, COR, BUN, and TP were determined, with TP being measured by a handheld optical refractometer.Rumen fluid was collected using an oral stomach tubing 4 h after cows had had their morning feed on d −15, −7, and 7 relative to parturition. The first (150 mL) was discarded to avoid saliva contamination. The rumen pH level was immediately measured, and 50 mL of each sample was immediately stored at −20°C for further analysis. Samples were analyzed for total volatile fatty acids (VFA), proportions of acetate, propionate, butyrate. valerate, isobutyrate, isovalerate, and ammonia.Ten cows per treatment were chosen for analysis purposes. All statistical analyses were performed using MIXED procedure of SAS . Continuous variables with repeated measurements including lying and ruminating behavior, the plasma and ruminal fermentation parameters were analyzed with the fixed effects of treatment, day of measurement, interaction between treatment and day of measurement, and the random effect of cow or calf nested within treatment. Variables with a single measurement during the study, such as milk production and composition, average calving date and colostrum quality were analyzed with the fixed effects of treatment of cow and the random effect of cow nested within treatment. It should be noted that feed intake data is excluded due to the technical issues experienced with the feeder. All treatment results are reported as least squares means. Significant differences were declared at p≤0.05 and tendencies at p≤0.10.The behavioral results of lying and ruminating are reported in The average yield, composition and quality of colostrum and milk are listed in The effect of stocking density on blood parameters in transition cows  and calvThe proportions of total VFA, acetate, propionate, butyrate, valerate, Isobutyrate, and isovalerate concentrations as well as acetate/propionate ratio, ammonia nitrogen and pH are reported in The BW, body length, withers height, breast circumference and abdominal circumference are shown in In this study, the results indicated that lying and rumination behavior can be affected by stocking density in the last 21 days of pregnancy of Holstein cows. Specifically, understocked cows (80% group) spent more time lying and ruminating compared to the other two groups (100% and 120% groups). This was more evident from d −21 to d −7. However, the lying time was similar at d −7 to d 0. These results are in agreement with a previous study , which cIncreased stocking density can also result in competition for available space  due to aHigher stocking density before parturition has been linked with decreased milk yield in the first 85 days of lactation . HoweverDuring the transition from late gestation to lactation, changes in the endocrinal system as well as feed intake are expected, which can affect metabolic status. By around 10 days before parturition, the NEFA concentration begins to rise in the blood irrespective of the decrease in dry matter intake . In an aUnder non-inflammatory conditions Ca is under hor monal control . We founFor successful passive immunity in calves, the serum TP should exceed 5.2 g/dL 24 h after colostrum feeding. All calves had greater than 5.4 g/dL of TP, indicative of successful passive transfer of immunity. The COR is usually high in newborn calves, and decreases with age as observed in this study. The Ca and BUN were within concentrations reported elsewhere for 1 week old calves.The BW and structural measurements of calves were also similar between treatments in this study. Similar BW of new born calves were reported in another study of cows under different stocking densities simulated by introducing new animals to the experimental group . The calThe VFA concentration and pH level were not different between groups at the selected days of sampling. However, the proportion of acetate, valerate, isobutyrate isovalerate changed with time. During the transition period, farmers often change animal diets from dry-off to a fresh diet. In our experiment, cows were introduced to a lactating diet which is high in concentrates compared to the dry-off diet. Hence, the observed decrease in acetate could be associated with the change in diet from a high to low-fiber diet. The observed levels in total VFA is likely a response to greater organic matter available for rumen fermentation . HoweverThis study demonstrated that prepartum stocking density affected the lying and ruminating time from d −21 to d −7 relative to parturition. Cows in the 80% stocking density group were associated with greater lying and ruminating times compared to the other groups. However, the differences were not significant enough to result in changes in the metabolic status a week before and after parturition. Furthermore, calf growth and performance were not affected in the first week of life.


Acetylated starch possessed higher solubility and swelling power than native starch under the temperature regimes considered. Addition of acetylated pigeon pea starch as a stabilizer in yoghurt had positive influence on the water holding capacity (7.7% to 10.4% compared to 13.3% in yoghurt stabilized with native pigeon pea starch) and whey syneresis  at the end of a 28-day cold storage period. In addition, pea starch-stabilized yoghurt possessed an enhanced sensory attribute (firmness), and compared favourably with gelatin-stabilized yoghurt in terms of overall acceptability. Thus, acetylated pigeon pea starch exhibited improved physicochemical properties and showed usefulness as a stabilizer in yoghurt because it enhanced the physicochemical, storability, and sensorial quality, while improving the body and texture of the product.The behaviour of graded acetylated pigeon pea starch during heat processing was evaluated in addition to the corresponding effect of their incorporation at 1.5% ( Yoghurt is a product of the lactic acid fermentation of milk and it is among the most common fermented dairy product widely consumed in many parts of the world. Syneresis (expulsion of whey) is a common technological defect in cultured semi-solid products, especially during refrigerated storage . ControlNative starches have some limitations preventing their use for industrial applications; these limitations can be overcome by techniques developed to modify, and thus, improve the physicochemical properties of starch. Modified food starches, specifically from cassava and corn, have been used as stabilizers in food systems to confer specific functions, such as increased viscosity, improved mouthfeel and creaminess, and reduced syneresis of yoghurt ,9. SeverCajanus cajan) is a legume that forms a good portion of the human diet in many African, Asian, and South American countries [Pigeon pea , PPAS2 (acetylated pigeon pea starch DS 0.09), and PPAS3 (acetylated pigeon pea starch DS 0.14) were obtained and used in this study. Starter culture DANISCO Yo Mix 495 LYO (containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was obtained from the Dairy unit, University of Manitoba, Winnipeg, Manitoba, Canada. Commercial homogenized and pasteurized milk  was purchased from Maizube farms, Minna, Niger State, Nigeria; skimmed milk powder (SMP) and sucrose were purchased from Ceci Supermarkets, Alagbaka, Akure, Ondo State, Nigeria. All reagents were of analytical grade and procured from Fisher Scientific  and Sigma Chemicals .Native and acetylated pigeon pea starches obtained from a previous study  in our lw/w) of starch were prepared in a flask, heated to 50, 60, 70, 80, and 90 °C for 30 min, with shaking every 5 min, and left for cooling to room temperature, thereafter, being centrifuged for 15 min at 3000× g. The supernatant was decanted, and the residual volume was determined. The solid part was dried in an oven for 2 h at 130 °C.The solubility and swelling power of starch was determined, following the method of Gani et al. . Suspensw/v db) was heated in a water bath at 90 °C for 30 min with constant agitation to ensure complete pasting. The starch paste was cooled to room temperature (around 25 °C) and the light transmittance of pastes was measured at 650 nm. The samples were stored for 5 days at 4 °C in a refrigerator and percentage transmittance was determined every 24 h using a UV/visible spectrophotometer .Paste clarity/light transmittance of starch paste was determined, as described by Craig et al. . An aqueThe pasting property was determined using a Rapid Visco Analyser (RVA) , as described in AACC 44-31A . About 2w/v) skimmed milk powder (SMP); 2% (w/v) sucrose was added as sweetener, followed by addition of 1.5% (w/v) acetylated or native pigeon pea starch as the stabilizer, and then, mixed thoroughly. The mixture was pasteurized at 85 °C for 30 min, cooled to 40–43 °C, and starter culture  per 100 L) was added, then, distributed into sterile 250 mL containers to allow easy collection of sample for analysis. Fermentation was carried out at 40 °C for 6 h to develop proper acidity. Fermentation was terminated and the yoghurt samples were stored at 4 °C for 24 h prior to chemical analysis, sensory evaluation, and storage stability test. The prepared yoghurt was stored in the refrigerator (4 °C) for 28 days and samples (three independent containers of 250 mL) were analysed for each of the parameters evaluated at 7-day intervals throughout the refrigerated storage period. A commercial yoghurt sample was used as a control, while laboratory control was prepared with the above stated materials  and 0.2% gelatin as the stabilizer.The yoghurt was prepared in triplicate, as described by Cui et al. . Two litpH of the yoghurt was measured at 23 °C using a pH meter  after calibrating with fresh pH 4.0, 7.0, and 10.0 standard buffers.TA expressed as percent of lactic acid was determined using titrimetric method, as described by Schmidt et al. . YoghurtYoghurt samples were mixed thoroughly using a glass rod and about 1 g of sample was oven-dried for 2 h; the percentage Total Solids (% TS) was calculated using the equation below:g and 4 °C. The amount of supernatant whey was recorded. The percentage syneresis was calculated using the equation below:Syneresis of yoghurt samples was measured according to the modified method of Guzman-Gonzalez et al. . Approxig at 4 °C for 10 min. The supernatant was collected and weighed, and WHC was calculated according to the following equation:The water holding capacity (WHC) of yoghurt is defined as its ability to hold all or part of its own water. WHC of the samples was determined using method reported by the authors in reference . YoghurtAssessment of the sensory attributes was carried out as described by Soukoulis et al. . Thirty Data were generated in triplicate and subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Sciences (SPSS) V. 17.0 . The mean values were separated using Duncan’s Multiple Range Test (DMRT) at 95% confidence level.3) also had the highest starch solubility (5.7% at 50 °C to 34.6% at 90 °C). Similarly, acetylation increased the swelling power, which is a measure of the hydration capacity of the starch granules. The swelling power showed direct proportionality to temperature and degree of acetylation.The effect of temperature on solubility of native and acetylated pea starches was progressive A. The pe1, PPAS2, and PPAS3, respectively.The effect of acetylation and storage duration on paste clarity of pigeon pea starch is shown in p < 0.05) reduced PT (87.80 to 85.60 °C). Pasting time (Pt) ranged from 4.93 to 5.07 min. The acetylated starches required less time to form a paste when compared to the native starch. Peak viscosity (Pv) ranged from 314.70 to 588.70 RVU and values reduced with an increase in level of acetylation. Fv of starches followed similar trend as Pv and Tv with PPNS showing highest Fv and the value decreased with increase in DS of starch. Similar to other pasting parameters, native pea starch showed highest breakdown viscosity (BD) and setback (SB), which was significantly (p < 0.05) reduced by 24% to 49% and 38% to 44%, respectively, when the starch was acetylated.Pasting properties of pigeon pea starch as influenced by acetylation are summarized in p < 0.05) in percentage syneresis, with yoghurt stabilized with native pea starch (YoPN) exuding the highest amount of liquid from the yoghurt matrix, thus, having the highest degree of syneresis (46%). The degree of substitution showed an inversely proportional relationship with degree of syneresis; the yoghurts containing acetylated pea starch had lower syneresis (18.6% to 22%) compared to YoCN and YoPN. However, the market yoghurt (CTR) had a far lower level of syneresis at 10.8%.Acetylated pigeon pea starch was employed as the stabilizer in set-type yoghurt and the physicochemical properties presented in Water holding capacity (WHC) and whey syneresis of the yoghurt during the 28 days was evaluated at 7-day intervals throughout the refrigerated storage period A,B. Commp > 0.05) for colour (7.1 to 7.6), whereas firmness of the yoghurt was significantly different (p < 0.05) from one another (5.8 to 7.2).The sensory attributes of the laboratory control (gelatin-stabilized yoghurt) compared favourably with the market control , except for aroma, flavour, and overall acceptability . GeneralIncreased solubility in acetylated starches can be attributed to the increased hydrophilicity of the starch . SolubilThe swelling behaviour of starch is dependent on the amylose content, as well as the presence of amylopectin and non-carbohydrate substances, especially lipids, which may inhibit swelling. Swelling commenced at 60 °C earlier than observed in native starch and the highest swelling capacity was observed at 90 °C for all starches (2.5 to 4.0 g/g), which indicates that the penetrating power of water into starch granules can be increased at high temperature. The increase in swelling power may be a result of starch gelatinization, which causes disruption of the granular membrane and weakening of the internal starch structure that favoured increased water absorption. Acetylation conferred a 1.5 to 2.1-fold increase in swelling of starch paste, an effect that progressed with temperature. Introduction of the bulky acetyl group promotes structural reorganization, resulting in repulsion between starch molecules, which facilitates increased water percolation within amorphous regions of starch granules with a consequent increase in swelling capacity .The high solubility and swelling capacities of acetylated pigeon pea starch may also be due to the introduction of hydrophilic substituting groups that allows the retention of water molecules, owing to their ability to form hydrogen bonds. This ensures a high retention of the water that enters the granule, thereby increasing the swelling capacity. This is a useful property in the manufacture of some confectionery products. A similar trend showing increased swelling capacity has been reported for chickpea  and blac2 and PPAS3 exhibited higher light transmittance than PPNS and PPAS1. More so, PPNS and PPAS1 displayed significant decreases in light transmittance up until the fifth day, unlike PPAS2 and PPAS3 that exhibited lower rates of decrease in light transmittance during the storage period. Low light transmittance observed in native pigeon pea starch may be a consequence of turbidity development during storage caused by granule swelling and intermolecular bonding that enhanced retrogradation of amylose chains [PPASe chains . In addie chains . Higher e chains . Substite chains , where ae chains  also repp < 0.05) reduced PT (87.80 to 85.60 °C). The high pasting temperature observed in PPNS indicate native pigeon pea starch granules exhibited higher resistance to swelling and membrane rupture, while lower pasting temperature in acetylated starches may be as a result of the introduction of the acetyl group to the amorphous region, which made swelling of the acetylated starch easier than in the native starch. The decreased pasting temperature may be a result of weakening of starch structure due to interrupted hydrogen bonds upon introduction of the acetyl group. This property is important in systems where thickening of gels is required at low temperature in order to reduce energy cost during production [Pasting temperature (PT) is an important indicator of the minimum temperature required to achieve cooking in the starch sample and depicts the temperature at which viscosity begins to increase during the heating process. In this study, PT ranged from 86 to 90 °C. PPNS had the highest pasting temperature, however, acetylation significantly  is a measure of the cooking time  and thisPeak viscosity is the maximum viscosity developed during or soon after the heating portion of the pasting period and it correlates to the final product quality. Generally, high viscosity of starch is desirable for industrial uses, for which a high thickening potential at high temperature is required and a high peak viscosity suggests more starch has been gelatinized during processing. The peak viscosity of native pigeon pea starch was higher than the modified counterpart, suggesting that the starch can be incorporated into food that requires high thickening such as custard, while lower viscosity of the acetylated starches could facilitate better flow of starch paste when introduced into food systems. Similar to the present finding, Wani et al.  reportedFinal viscosity (Fv) is a parameter used to indicate the ability of starch paste to form a viscous paste resultant from retrogradation of soluble amylose during cooling and gelling of amylose . High Fvp < 0.05) reduced this property by 24% to 49%. High BD infers less stability of starch during heating. This property was lower in acetylated starches, which suggests that they may exhibit better starch granule swelling with slow tendency to lose viscosity and might be able to withstand more heating and shear stress [3, will be more stable under high temperature processing or storage conditions than PPNS. High BD values reflect granular swelling that make starch granules more susceptible to shear [Breakdown (BD) measures the tendency of swollen starch granules to rupture when held at higher temperatures and subjected to continuous shearing; this is also indicative of the stability of the starch upon heating as well as a measure of the susceptibility of cooked paste to disintegration. Highest breakdown viscosity (BD) was observed in PPNS; acetylation, however, significantly  lower in the acetylated starches (202.4 to 222.80 RVU) compared to the native pigeon pea starch counterpart. Ashogbon [Set back (SB) of the starches was significantly  between them. This suggests that incorporation of pea starch had no significant effect on pH.Acidity of yoghurt is a result of fermentation of milk sugar (lactose) into lactic acid by the starter culture during incubation. The development of lactic acid in fermented dairy products is essential for the proper formation of a yoghurt gel network. Acidity is inversely related to pH value, as increased lactic acid production results in a corresponding pH drop. Nevertheless, pH is a valuable tool for measuring the extent of lactic acid fermentation, since acidity influences the perception of other attributes  and playThe acidity of commercial yoghurt (0.90%) was significantly different from those of developed samples (1.03% to 1.21%), and this may be due to differences in starter culture, milk type, fermentation time, and temperature. Acidity observed for LCTR was within the range (0.98% to 1.10%) in another report for gelatin-stabilized camel milk yoghurt , which mSolids content was higher in yoghurt with stabilizer  than the YoCN, whereas graded acetylation of pigeon pea starch showed no effect on the solid content of yoghurt. The high total solids content supports the firm, custard-like body of set-type yoghurt; values are significantly higher than the 13% to 17% range earlier reported by Smit  for comm3 in terms of retrogradation tendency, breakdown, and set back. The improved properties of PPAS3 may have contributed to the improved ability to bind the free water as well entrap the yoghurt gel during the coagulation process. Significant whey syneresis (46.5%) observed in YoCN may be attributed to the absence of a stabilizer and, hence, the expulsion of liquid could not be efficiently prevented. The yoghurt stabilized with acetylated starches had lower syneresis values (19–22%), although was significantly higher than the gelatin-stabilized yoghurt and market sample. However, acetylated starch with higher DS (PPAS2 and PPAS3) resulted in reduced whey expulsion when compared to 36.8% in the sample stabilized with native starch (YoPN). Overall, starch-stabilized yoghurts showed 53–60% reductions in whey syneresis when compared to control yoghurt without pea starch (YoCN). Syneresis is an important factor in set-type yoghurt, as it can lead to the accumulation of whey on the surface of the yoghurt, which can result in poor consumer acceptance [Syneresis ranged from 10.8% to 46.5%, and the market sample (CTR) exhibited the lowest value; this property significantly differed among the yoghurt samples. The loss of ability of the yoghurt gel to entrap the serum phase due to the weak gel network results in whey separation, which is also referred to as syneresis . The perceptance ; the prep < 0.05) higher than yoghurt without pigeon pea starch that had 57% WHC at day 0. More so, the WHC of YoPA1, YoPA2, and YoPA3 was not significantly different (p > 0.05) from each other and compared with no significance difference with LCTR. There were no significant changes in the WHC of each yoghurt sample during storage, except on the 28th day, where lower WHC for some of the samples was observed. The results suggest that introduction of acetylated pea starches as stabilizers was able to effectively bind water, thus, reducing water loss from the yoghurt gel matrix.The DS of the pea starch showed no significant effect on the WHC of yoghurt; however, native pea starch exhibited lower WHC (66%), which gradually reduced over the 28-day cold storage period. The WHC (85%) of yoghurts with stabilizer (acetylated starches) was significantly  showed no significant difference (p > 0.05) in syneresis percentage. Although the acetylated pigeon pea starches did not totally prevent serum separation during storage, they sufficiently reduced syneresis to levels that are similar to that of the commercial sample.The stabilized yoghurts showed no significant susceptibility to syneresis over the refrigerated storage period when compared to yoghurts with no stabilizer (YoCN) or with native starch (YoPN), which was initially stable for 14 days, after which progressive syneresis was observed. Nonetheless, YoPN had a lower degree of serum separation than YoCN, suggesting that native starch exhibited better ability to retain water within the yoghurt matrix than samples without stabilizer. Degree of substitution had no significant effect on the amount of syneresis, as yoghurts with the acetylated starches (YoPAp > 0.05) in their sensory attributes, as shown by the similar values obtained for YoPA1, YoPA2, and YoPA3. Moreover, the colour of all yoghurt samples was not significantly different (p > 0.05) from the commercial sample, thus, addition of pigeon pea starch had no effect on the colour quality. The introduction of PPAS3 in yoghurt showed the best stabilizing effect by enhancing firmness of the sample (YoPA3); regardless, this sample did not significantly differ (p > 0.05) from the others, except YoCN, which may result from the absence of any form of stabilizing agent in the product.The sensory attributes of the laboratory control (gelatin-stabilized yoghurt) compared favourably with the market control  except for aroma, flavour, and overall acceptability ; this ma3 also showed superior properties. The sensory ratings showed that the use of native starch was inadequate for yoghurt stabilization because YoPN exhibited poor firmness and high syneresis. YoPA3 had the highest overall acceptability among the experimental yoghurt samples, which might be attributed to the superior firmness. The lower level of syneresis may have contributed to the increased firmness of YoPA3, despite the lower viscosity of the modified starch ingredient. This is because texture  is one of the major determinants of yoghurt quality based on its influence on yoghurt appearance, mouthfeel, and overall acceptability [The sensory results correlate to the WHC and syneresis, where YoPAtability . Earliertability . Similartability  reportedThe higher sensory scores for firmness, consistency, syneresis, and overall acceptability showed that the use of acetylated pigeon pea starch as a yoghurt structure stabilizer has greater potential than the native starch with respect to adoption by the industry as a food ingredient.3 with 0.14 DS produced the most acceptable sample and could be considered a potential ingredient for industrial production of set-type yoghurt.Acetylation of pigeon pea starch enhanced the physicochemical properties , which promoted its suitability as a functional ingredient in yoghurt formulation. The use of acetylated starch as a stabilizer was comparable with gelatin and suitable for significant reductions in serum separation, while maintaining firmness, consistency, flavour, and overall acceptability of yoghurt. PPAS


Butyrivibrio fibrisolvens. The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes.Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium  Despite the abundance of viruses in the rumen, little is known about the bacteriophage (phage) population in the rumen microbiome compared to other predominant microbes in this environment . The infSiphoviridae family; two that target the bacterial host Prevotella ruminicola and one which targets Streptococcus bovis, and another two belonging to the Podoviridae family that target Ruminococcus albus 2SO4, 6.0 g/L; NaCl, 6.0 g/L; MgSO4⋅7H2O, 0.6 g/L; CaCl2⋅2H2O, 0.6 g/L.2 medium ; using 1ad libitum on grass, with ∼300 g sugar beet and grass nuts supplemented each morning. Rumen fluid samples were obtained in the same way from three 10-year-old Holstein-Friesian dry cows which were grazing ad libitum on grass, with ∼500 g dairy concentrates in the morning during sampling. Rumen fluid was obtained under the authority of licenses under the United Kingdom Animal Scientific Procedures act 1986. All animal associated research is managed according to the Aberystwyth University Animal Welfare and Ethics Review Board1. Each of the sheep rumen fluid samples were squeezed and strained through a sieve and then combined into one air-tight pre-warmed container. The same procedure was carried out for the cow rumen fluid and collected in another air-tight pre-warmed container.Samples of rumen fluid and feces were obtained from fistulated sheep and cows. Hand grabs of rumen contents were obtained through the cannulae of three 7-year-old Aberdale cross Texel sheep which were grazing Feces were caught in a gloved hand once excreted naturally from three sheep of the same flock and transferred to sterile plastic bags. Cow feces were obtained soon after excretion; a small amount of fresh fecal matter was taken from the field, collected into pots, and transferred into sterile centrifuge tubes. Three fecal samples each for cows and sheep were collected this way and screened for phage presence. Where necessary, fecal samples were refrigerated for less than 1 week before use to avoid a freeze/thaw cycle if stored in a freezer.g for 15 min at 4°C, and the supernatants stored on ice and filtered through 0.45 μm pore size low-protein binding PES syringe filters and retained on ice. Around 10 g of feces were weighed out into centrifuge tubes and a volume (in ml) equivalent to the mass (in g) of the sample of Phage Storage Buffer  in the anaerobic cabinet with gas mix of 10:10:80 of CO2, H2 and N2  (A lawn of host bacteria was achieved by adding 1 ml of overnight (or actively growing) culture of entific) . Once seUpon an initial indication of phage activity in a sample, then in later stages to purify and propagate positive phage samples, a plaque assay was carried out. An aliquot of 10 μl of the sample positive for phage activity was combined with 1 ml of host bacterial culture, left to incubate for no more than 15 min, mixed with 3 ml of soft agar [0.8% (w/v)] and poured over bottom agar [1.5% (w/v) M2 agar] . Plaquesg for 2 min to pellet any agar, and the supernatant was filtered through a 0.45 μm pore size PES syringe filter. This resulted in five phage samples, temporarily termed phage C, D, J, M, and P.Once the plaque morphology was believed to be homogenous, or at least three rounds of purification had taken place, the phage was eluted from a confluent plaque assay plate by adding 5 ml of PSB to the plate, macerating the agar gently using a spreader, and leaving to incubate at room temperature on a rocker, for at least 30 min. The eluate was aspirated using a pipette tip into a microcentrifuge tube, spun at 15,000 × g for 10 min, and filtering the supernatant through a 0.45 μm pore size PES syringe filter. Of this, 600 μl was precipitated overnight with PEG/NaCl, as well as the negative control. From each of these controls, 40 μl also underwent DNA extraction alongside the phage samples. The FastDNATM Spin Kit for Soil  was used to extract DNA from the lysate samples, using the proprietary FastPrep-24 for 30 s at 6.0 m/s, and spinning at 14,000 × g for 5 min at 18°C. DNA was eluted from the column with 30 μl of supplied DES water. The DNA concentration was determined using the Qubit fluorometer  and the high sensitivity DNA assay. Those samples that had a low concentration were increased using a DNA Speed Vac , or in the case of phage sample J, was re-extracted using 900 μl of phage eluate and combining this with 300 μl of the supplied sodium phosphate buffer in the first step. DNA was eluted from the column first with 30 μl of DES water, then a second time with 50 μl. The concentration of DNA in the re-extracted and concentrated sample were measured again, and all samples were diluted with nuclease free water to achieve a final concentration of ∼2 ng/μl in 10 μl necessary for sequencing. Quality of the DNA was tested using spectrophotometry and the 260/280 ratio  and sequencing was done in-house, where the isolated phage DNA was first diluted to 1 ng/ul, and libraries then prepared using the Illumina NextEra XT protocol as per the manufacturer’s instructions (selecting AMPure bead ratio as suggested for 2 × 300 bp reads). Libraries were quantified via Qubit fluorescence spectrophotometry, pooled at an equimolar ratio, and diluted to 6 pM before loading on an Illumina MiSeq platform using a v3 600-cycle kit in 2 × 300 bp format. The five samples are available on NCBI as BioSamples under the BioProject accession code PRJNA613207, and raw reads for these in the Sequence Read Archive (SRA) under the accession code SRP255162.Phages from 800 μl of eluates were precipitated using PEG/NaCl overnight and resuspended in a volume one tenth of the initial volume, 40 μl of which was then used for nucleic acid extraction. Controls were added to the nucleic acid extraction process to evaluate the extraction process and other downstream applications, such as gel electrophoresis. The controls were comprised of molecular grade water as the negative control, and a “host lysate sample” as a positive control, which was made by taking an aliquot of host bacterial liquid culture, vortexing briefly and boiling at 100°C for 15 min to lyse the cells, before spinning at 15000 × 2 and repeat regions were identified. A 127 bp length of sequence in each of the genomes was repeated as an overlap of the assembled start and end (as the genomes were circular), which corresponded to the k-mer size used by SPAdes during assembly, and was removed. BWA-MEM v0.7.16a-r11810  nucleotide collection (nr/nt) using blastn  and defa4, Prodigal v2.6.3 with default settings with output as GFF (Open reading frames (ORFs) were then predicted using Glimmer v3.02  with deft as GFF  and PHANt as GFF  with def–5  database supplied with multiPhATE v1.0  for each ORF was also noted. Where gene callers did not agree on an ORF, the ORF with the best hit was used, considering coverage of query and subject and percentage identity to determine the best.Nucleotide and protein sequences for ORFs in all four categories were obtained using gff2bed tool , getfas–5  and quer–5  were retATE v1.0 . Protein9, TIGRfam10  and the GC content and codon usage were summarized in Geneious Prime. Additionally, the presence of prophages was predicted using the PHASTER online tool17  with the highest coverage was 100% similar at the nucleotide level to the phage genome reconstructed in phage C sample, as determined by ANI. This genome did not undergo further analysis. The whole phage genomes were queried in the NCBI virus nucleotide database and revealed that there was no significant similarity to viral genomes published and deposited in this database previously. Further searches against the viral representative reference sequence database and viral non-redundant RefSeq database from NCBI also did not reveal similarity to any previously deposited viruses. Therefore, as it is likely that these five phage genomes are novel, they have been named; phage sample C yielded Butyrivibrio phage Bo-Finn, phage sample D yielded Butyrivibrio phage Arawn, phage sample J-2 yielded Butyrivibrio phage Idris and phage sample M yielded Butyrivibrio phage Ceridwen. The summary of the samples, the source of the samples, the genome contigs reconstructed and subsequent names can be seen in To determine the similarity between these phage genomes, genome comparisons were first carried out using Mauve , then byThe reconstructed phage genomes ranged from 31 (Arawn) to 39.7 Kbp (Ceridwen) in length , with nuSiphoviridae; tail terminator, major tail protein, two chaperones, tape measure protein (TMP), distal tail protein, tail-associated lysozyme and baseplate/tip proteins, which together form a functional ‘tail morphogenesis module’  . The two module’ . Figure oviridae , and thein vitro and phage Arian harbors an endolysin (QHJ73651.1), and phage Bo-Finn an endolysin (QHJ73713.1) and putative holin (QHJ73715.1). Moreover, a tRNA gene for the amino acid glutamine was predicted in these genomes, which is used in 3.6% of coding genes in genome of phage Arian and 4.4% in Bo-Finn, compared to 2.7% of coding genes in the bacterial host genome . The two genomes are syntenous, with highly similar genome sequences at the nucleotide level. The most noticeable difference between the two genomes is at 21.3 Kbp, where the similarity is low, and a different sequence in phage Arawn results in three ORFs predicted in this region . The sere family . Unlike oviridae , and thein vitro undergoing the lytic cycle, the presence of these genes suggests the potential for Butyrivibrio phages Arawn and Idris to undergo the temperate lifecycle. The fully annotated genomes of phage Arawn and Idris can be seen in Both phage Arawn and Idris were predicted confidently by PHACTS to undergo the temperate lifecycle. The major capsid proteins are similar to those in the temperate phages Streptococcus phage phiD12 and Streptococcus phage phi-SsUD.1 . A putatAs with phages Arian and Bo-Finn, a blastn search for the entire genome sequences of phage Arawn and Idris against the NCBI virus genome database did not reveal any significant alignments covering more than 8% of the query genome.Siphoviridae family, and one of Podoviridae. Therefore, it is likely that this phage belongs to the Siphoviridae family.The longest of the Butyrivibrio phage genomes belongs to phage Ceridwen, which also has the densest genome, with more predicted ORFs in the genome proportional to the length . Of thesin vitro.Phage Ceridwen was predicted non-confidently as having a lytic lifecycle by PHACTS, but the presence of an ORF similar to an antirepressor (QHJ73772.1) suggests lysogenic abilities. This antirepressor may act on the gene upstream of it; a gene homologous to the XRE family of translational inhibitors (QHJ73771.1). The most characterized phage repressors belong to this family , which aA blastn search for the entire genome sequences of phage Ceridwen against the NCBI virus genome database did not reveal any significant alignments covering more than 1% of the query genome.in vitro to undergo the lytic lifecycle, infecting the same B. fibrisolvens host strain. The phages likely belong to the Siphoviridae family, as evidenced through genes encoded, genome organization, size, and similarity to other phages belonging to this family. All five phages possess genomes with sizes > 20 Kbp and <125 Kbp, and tail morphogenesis modules typical of siphoviruses , which does agree with the observation made previously that shorter phage genomes tend to have higher GC contents . There iSiphoviridae that were sequenced previously , Arawn (MN882550), Idris (MN882554), Ceridwen (MN882553), and Arian (MN882551).The datasets generated for this study can be found in the BioProject Accession The animal study was reviewed and approved by Aberystwyth University Animal Welfare and Ethics Review Board.JF, AK-S, and CC conceived the project. JF carried out experimental work and analysis with support from JP, AK-S, CC, DR, and AC. All authors contributed to manuscript writing and editing.DR was employed by Dynamic Extractions Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, LDPE samples were prepared by melt blending with different cooling processes, which were natural air cooling, rapid air cooling, water cooling and oil cooling, respectively. According to polarization microscope (PLM) and differential scanning calorimeter (DSC) tests of these four low-density polyethylene (LDPE) samples, the effect of different cooling processes on polythene crystalline morphology could be studied. According to conductivity, dielectric frequency spectra and space charge tests, the effect of crystalline morphology on dielectric macroscopic properties could be explored. The microstructure characteristic results indicated the cooling medium significantly affected polythene crystalline morphology. When the samples were produced with natural air cooling, the crystalline grain size was large. On the other hand, after rapid air cooling, water cooling and oil cooling processes, the samples’ crystalline grain dispersed uniformly, and the grain sizes were lower. The space charge testing results indicate the samples produced with water cooling and oil cooling processes restrained the electrode injection in the process of pressurization. During short-circuits, the rates of charge release of these two samples were fast, and the remaining space charges were fewer. The conductivity and dielectric frequency spectra testing results indicated the conductivities of samples produced with water cooling and oil cooling processes were both less than those of samples produced with a natural air cooling process. Besides, with increasing experimental frequency, the relative dielectric constants of all testing samples decreased. Among them, the relative dielectric constant of the LDPE sample with the natural air cooling process was the largest. However, the crystalline structures of samples produced with rapid air cooling and water cooling processes were close, which restrained the movement of polymer macromolecule chains. Thus, the dielectric constants were lower. Additionally, because of the influence of relaxation polarization and dipole polarization, the dielectric losses of LDPE with water cooling and oil cooling processes increased to varying degrees. With rapid economic development, there has been an ever-growing demand for electrical energy. New types of power transmission technology, such as high-voltage direct current transmission (HVDCT) and flexible AC transmission (FACT), have emerged. On the other hand, insulation materials’ aging caused by the space charge under DC high voltage (DCHV) is serious ,2,3,4. BIn this article, low-density polythene (LDPE) insulation material was prepared by melt blending and LDPE sheeting samples were pressed by a plate vulcanizing press machine using different methods. These were natural air cooling, rapid air cooling, water cooling and oil cooling. According to the PLM and DSC tests, the samples’ crystalline morphology was observed, from which the effects of different cooling media on materials’ crystalline morphology were analyzed. According to pulsed electroacoustics, the space charge accumulation and attenuation of each sample under pressure and their short-circuit conditions could be measured, from which the different cooling medias’ effects on polythene space charge behavior could be explored. The volt-ampere characteristic testing equipment was built based on picoammeters. Thus, the relationship between samples’ conductivity and field strength was measured, from which the effects of different cooling media on materials’ conductivity were analyzed. The dielectric spectra of polythene and its nanocomposites were also measured. The dielectric constant and loss factor change with frequency were recorded, from which the effects of different cooling media on the dielectric constant and loss factor could be explored.In sample preparation, firstly, the LDPE particles were added into the torque rheometer, the temperature of which was set to 150 °C. When the particles achieved the melting state, these samples were pressed into sheets by plate vulcanizing press machine . In order to explore the effects of different cooling media on the crystalline morphology and dielectric properties of LDPE, these samples were cooled down to room temperature with different cooling media. The samples’ thicknesses were 100 μm, 200 μm and 300 μm respectively, and all of them were dealt with PLM test, DSC test, dielectric frequency spectra test, conductivity test and space charges test.Natural air cooling: After melt pressing for 15 min with a 150 °C plate vulcanizing press machine, the samples were taken out and placed into another plate vulcanizing press machine at room temperature. Then, the 10 MPa pressure was maintained until the samples were cooled down to room temperature.Rapid air cooling: After melt pressing, the samples were taken out of the 150 °C plate vulcanizing press machine and exposed to the air. Then the samples were processed with rapid cooling by an electric fan.Water cooling: After melt pressing, the samples were taken out of the 150 °C plate vulcanizing press machine and put into warm water rapidly for cooling. All these samples were then placed in air and dried.Oil cooling: After melt pressing, the samples were taken out of the 150 °C plate vulcanizing press machine and put into cable oil rapidly for cooling. All these samples were then placed in air and dried.In the cooling process, the LDPE samples were cooled by the processes of natural air cooling, rapid air cooling, water cooling and oil cooling respectively. The specific implementation process was as follows:After the cooling process, all the samples were place in a drying tank for the relevant test. The samples prepared with natural air cooling, rapid air cooling, water cooling and oil cooling were marked A1, A2, W3 and O4.According to the PLM test, the crystalline morphology and crystal sizes of different samples could be observed. The test samples’ thickness was 100 μm. These samples surfaces were corroded with a blend of potassium permanganate and concentrated sulfuric acid. After ultrasonic cleaning, the samples were placed on a glass slide and LDPE crystallization was observed by PLM.−1. The enthalpy change during the heating process was recorded, from which the materials melting peak temperature Tm and melting enthalpy ΔHm were obtained. These samples were then cooled down to 25 °C at the same speed. According to the enthalpy change in the cooling process, the crystallization peak temperature Tc and half wide of exothermic crystallization peak ΔTc were obtained. Combining the parameters above and Equation (1), the different samples’ crystallinities were calculated.The isothermal crystallization and melting process of the LDPE polymer were observed by a DSC test. The test samples’ thickness was 100 μm. Under 150 mL/min drying nitrogen, the samples were heated to 140 °C at a uniform speed of 10 °C·minIn the conductivity test, the different samples’ conductivities were tested with a picoammeter. The test samples’ thickness was 200 μm. Ten measurements per sample were made, then the results were averaged. The boost method was step-up. The aluminum three-electrode system, of which the diameter was 50 mm, was evaporated on the samples’ surface by a vacuum coating machine. The samples’ conductivity could be calculated by Equation (2) .(2)σ=IUσ is the conductivity; U is the test voltage; d is the samples’ thickness; D1 is the inner electrode diameter on the samples’ upper surface; and g is the gap distance, which was 1 mm.In Equation (2), 5 Hz. The aluminum electrode system, of which the diameter was 25 mm, was evaporated on the samples’ upper and lower surface by a vacuum coating machine. In order to eliminate the effect of moisture and residual charges, the samples were placed in an 80 °C oven and treated with a short circuit for 24 h. Under room temperature, the relative dielectric constant and the loss factor of different samples were measured.In this article, the broadband dielectric spectra analyzer was used for the dielectric frequency spectra test. The test samples’ thickness and diameter were 100 μm and 40 mm, respectively, and the experimental frequency range was 1–10Pulsed electroacoustics was used for the space charge distribution test. The test samples’ thickness and diameter were 300 μm and 80 mm, respectively. Under the 10 kV/mm, 20 kV/mm and 40 kV/mm applied field strengths for 20 min pressure and short-circuit treatment for 30 min, the space charge distributions of the different samples were observed.The PLM patterns of samples prepared with different cooling mediums are shown in The crystalline morphology of sample A1 is shown in Hm and melting peak temperature were obtained. From the cooling curve, the crystallization peak temperature Tc and polymer crystallization rate ΔTc were obtained. The isothermal crystallization and melting process parameters of the different samples are shown in The heating and cooling process curves of different samples are shown in Tm, of samples A2, W3 and O4 were all lower than that of sample A1. On the other hand, the crystallization peak of sample A1 was the lowest. This illustrates that the samples W3 and O4 crystallized at a high temperature. Among these, the crystallization peak Tc of sample O4 was the highest, and the half-wide exothermic crystallization peak ΔTc was the lowest. ΔTc was the polymer crystallization rate, so sample O4 possessed a fast crystallization rate, high crystallization temperature and perfect crystalline morphology. The samples W3 and A2 took second place. According to the DSC test results, the crystalline morphology and crystal sizes of the four samples in PLM patterns were verified further. The different cooling methods and media may have affected the polymers’ crystalline morphology.Combining the test results for The curves of different LDPE samples’ conductivity changing and field strength are shown in From The different cooling mediums had some effect on LDPE conductivity, and the conductivities under high field strength and low field strength were different. In order to obtain a better comparison, conductivity line charts and bar charts for different cooling samples under 10 kV/mm and 50 kV/mm electric fields were drawn, which are shown in This was because the electrodes could not inject charges into the LDPE under a low electric field. The ions played a key role in electric conduction, so it was ionic conduction. In the preparation of different samples, more impurity ions were introduced, which increased the carriers’ concentration. Therefore, the conductivity also increased. Under a high electric field, the electrodes injected more electrons and holes. The electrons, holes and impurity ions all participated in electric conduction. Among them, the electrons played the key role, so it was electronic conduction . With a The different cooling processes changed the polythene microstructure, which also affected the polythene polarization. At the same time, the samples’ dielectric constants and loss were changed. Therefore, the effect of the cooling process on dielectric constant and dissipation factor of LDPE could be explored by a dielectric frequency spectra test. The changing relationships between the dielectric constant and dielectric loss for different samples with frequency are shown in 5 Hz), a small part of polarization could not keep up with the applied electric field change, so polarization could not be established. In addition, the relative dielectric constant decreased slightly [From slightly ,28. AmonFrom The space charge distributions of different samples are shown in From The space charge distribution of four samples during the short-circuit moments of 10 s, 900 s and 1800 s are shown in From From the PLM and DSC test results, the polymers’ crystalline morphology was greatly affected by cooling medium and cooling rate. The grain size order of the four samples was as follows: O4 < W3 < A2 < A1. At the same time, in sample O4, the grains dispersed uniformly. Besides, sample O4 possessed a fast crystallization rate, high crystallization temperature and perfect crystalline morphology.From the macroscopic test results, under a low electric field, the four samples’ conductivity increased with rising field strength. Under a field strength of 50 kV/mm, the conductivity order of the four samples was as follows: O4 < W3 < A2 < A1. Additionally, with different field strengths applied for 20min, the space charge density in sample O4 was the least. After short-circuiting for 30min, the remaining space charge quantity in sample O4 was also the least.Sample O4 possessed a complete crystalline structure. In this material, the crystal size was small, and the crystallization rate was high. The space charge effect was obviously restrained. Under a high electric field, the conductivity was low. Therefore, crystalline structure changes could improve polymers’ dielectric properties. This provides a theoretical foundation for follow-up research about polymer composite insulating materials.For the purpose of changing the crystalline structure of the polymer matrix, in this article, different cooling media and cooling rates were used. Four LDPE samples were prepared with natural air cooling, rapid air cooling, water cooling and oil cooling. According to PLM and DSC tests, the effects of different cooling methods on polymers’ micro-crystalline morphologies could be explored. According to conductivity, dielectric frequency spectra and space charge tests, the influence mechanisms of micro-crystalline morphology on polymers’ dielectric properties were explored. Specific conclusions are as follows:


Introduction: Typhoid fever is a major health problem in developing countries. Extensively drug-resistant (XDR) typhoid is an emerging threat to world health. The objectives of this study were to report our blood culture proven patients having XDR typhoid and compare the rate of gastrointestinal (GI) and hepatobiliary manifestations and complications of antimicrobial sensitive and resistant strains.Salmonella typhi. A total of 57 cases of Salmonella were identified, of which 10 were nonresistant, seven multi drug-resistant (MDR), 39 extensively drug-resistant (XDR), and one was extended-spectrum beta lactamase (ESBL) positive. Alarmingly, one of the S. typhi isolate in addition to the first line drugs, was also resistant to azithromycin. Patients were treated with antibiotics according to antimicrobial susceptibility of the Salmonella in accordance with the World Health Organization (WHO) and Medical Microbiology and Infectious Diseases Society of Pakistan (MMIDSP) guidelines and GI and hepatobiliary complications were recorded.Materials and methods: This prospective observational study was carried out at a tertiary care hospital in Pakistan, from January 2019 till August 2020 on all consecutive blood culture proven patients of Results: Overall rate of complications was low. Some 10% (1/10) with nonresistant typhoid, 14% (1/7) with MDR, and 15% (6/39) of our patients with XDR typhoid fever had abdominal tenderness (p=0.95). None of the patients had GI bleeding, abdominal abscess, or peritonitis. Some 20% (2/10) patients with nonresistant typhoid, 29% (2/7) with MDR, and 18% (7/39) with XDR typhoid developed acute hepatitis, with greater than three times elevation of liver transaminases. There was no statistically significant difference in the occurrence of hepatitis between these groups (p=0.98). Interestingly, one of our patients with XDR typhoid also developed cholestatic hepatitis.Conclusion: There is no significant difference in GI and hepatobiliary complications amongst antimicrobial sensitive and resistant strains of typhoid. However, emergence of resistant strains calls for focus on prevention and judicious use of antimicrobials. Salmonella typhi (S. typhi). It continues to be a major health problem in the developing countries with 21.6 million cases causing about 250,000 deaths annually [S. typhi is the causative organism in as many as 30% of bacterial hematogenous infections in Asia [Typhoid fever is caused by Gram negative bacteria, In 2016, an outbreak started from Sindh, Pakistan of a drug-resistant strain that has further spread to other parts of the country and so far, more than 5200 patients of this extensively drug-resistant (XDR) strain have been reported. This strain remains resistant to the standard first line and second line agents used for treatment of enteric fever, including fluoroquinolones -5. PreseAntimicrobial resistance (AMR) is quite widespread, and patients who are treated with ineffective antimicrobials exhibit improper clinical response with excessive complications and high mortality. AMR also leads to prolonged fecal shedding of bacteria, which causes sustained spread in the community with emergence of secondary cases -10.The aims of this study were to report our blood culture proven patients of XDR typhoid and compare the rate of gastrointestinal (GI) and hepatobiliary manifestations and complications of antimicrobial sensitive and resistant strains.S. typhi. All cultures were processed on Mueller-Hilton agar at 37°C, in a single laboratory and antibiotic susceptibility was assessed by disc diffusion technique and zones of inhibition were elucidated in accordance with the Clinical and Laboratory Standard Institute (CLSI) recommendations [This prospective observational study was carried out at CMH Lahore Hospital, from January 2019 till August 2020 on all blood culture proven patients of ndations . MinimalThe study was conducted in line with principles of Helsinki’s declaration, after getting approval from the institution’s ethics committee. All eligible patients who were admitted as medical inpatients with fever were followed. Only those patients in whom culture confirmed growth of Salmonella were included in the study. Participant’s demographic, clinical, and therapeutic details were recorded during the course of admission in a standardized proforma, after getting informed consent. Data were analyzed through Statistical Package for the Social Sciences (SPSS) version 25 for Windows. Quantitative variables like age, duration of fever, and laboratory test results were presented as mean with or without standard deviation. Qualitative variables were shown as frequencies and percentages. Linear regression analysis was used to find correlation between different parameters. Chi-square was used to probe the association of complications between drug sensitive, multi drug-resistant (MDR), XDR, and ESBL positive strains of Salmonella and value of p ≤ 0.05 was considered to be statistically significant.Resistance patterns were assigned according to the World Health Organization (WHO) definitions, which were as follows .Nonresistant typhoid: Typhoid fever caused by S. typhi and/or paratyphi A, B, or C strains which are sensitive to first line drugs  and third generation cephalosporins, with or without resistance to second line agents (fluoroquinolones).Multi drug-resistant typhoid: Defined as resistant to three first line drugs used to treat typhoid - chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole, with or without resistance to second line agents.Extensively drug-resistant typhoid: Defined as resistant to first and second line agents as well as third generation cephalosporins.Medical Microbiology and Infectious Diseases Society of Pakistan (MMIDSP) has further added another category of ESBL positive typhoid based upon the culture isolates of Salmonella, which is defined as follows :ESBL positive typhoid: Typhoid fever caused by S. typhi strains which are resistant to third generation cephalosporins but may be sensitive to chloramphenicol, trimethoprim-sulfamethoxazole, or fluoroquinolones.Out of the 57 study participants, 45 (79%) were male while the mean age of the patients was 30.7 years. Demographic and baseline characteristics of the patients are shown in Table S. typhi isolates 10 were nonresistant, seven MDR, 39 XDR and one was ESBL positive, as shown in the bar chart below (Figure All patients had high grade fever on presentation with a mean duration of 13 ± 6.64 days. On admission, in all suspected patients of typhoid fever blood cultures were drawn and they were started on parenteral ceftriaxone 1 g every 12 hourly. In patients who did not show defervescence of fever on ceftriaxone after 48 hours, multiple further sets of blood as well as urine cultures were drawn. Once culture results were available, antimicrobials were switched according to the susceptibility results. All patients with XDR typhoid were treated with either alone or a combination of azithromycin and meropenem, according to MMIDSP guidelines [Table Typhoid is one of the most common enteric infections of the developing countries. The organism’s feco-oral mode of transmission and predilection for the GI tract makes it the most common site of complications. As many as 30%-45% of patients report abdominal pain . AssociaHepatomegaly is a common finding in patients with typhoid fever. It is associated with liver dysfunction, partly due to the effect of bacteria causing proliferation of the reticulo-endothelial cells and partly due to the effects of medications -21. ThisSpleen being a reticuloendothelial organ, is frequently involved in typhoid fever. Splenic involvement can vary from splenomegaly which is very common, to splenic abscess or rupture that are infrequent complications , 26. SplOther GI and abdominal complications that have been reported to be associated with typhoid fever include acalculus cholecystitis, Gram negative sepsis leading to gall bladder gangrene and perforation, concomitant gall bladder and ileal perforation, duodenal ulceration with polyserositis, ileal ulceration with hematochezia, abdominal abscess, ovarian abscess, pancreatitis leading to pancreatic abscess, mesenteric lymphadenitis, and ascites. None of these complications were observed in any of our patient with typhoid fever , 27-30.The XDR typhoid fever is an emerging threat to public health around the world. GI tract and hepatobiliary complications have a major share in the spectrum of dreadful complications that can be encountered in these patients. Effective treatment of these complications requires antibiotic treatment according to the organism’s antimicrobial susceptibility. Finding of a bacterial isolate which was resistant to azithromycin is a cause of great concern, as the antimicrobial options for XDR typhoid are already limited. Although we did not find any statistical difference in the occurrence of GI or hepatobiliary complications in the antibiotic sensitive versus antibiotic resistant groups, the reliance on antibiotics for successful treatment calls for public and healthcare awareness for prevention and judicious use of antimicrobials to minimize the emergence and spread of resistant isolates.


Oryza sativa L.) seedlings were grown hydroponically in iso-osmotic stress conditions developed from NaCl or polyethylene glycol (PEG). The effects of Si on the growth of shoot and root of rice under salinity and PEG-derived osmotic stress were evaluated and further compared using principal coordinate analysis (PCoA). We also analyzed the concentrations of Na, K, and compatible osmolytes, tissue sap osmotic potential, antioxidant enzymes activities, and the expression of aquaporin genes. Generally, Si significantly promoted shoot and root growth in rice exposed to both NaCl and PEG. PCoA shows that the Si-induced distance change under NaCl treatment was larger than that under PEG treatment in the shoot, while the Si-induced distance changes under NaCl and PEG treatments were at an equal level in the root. Under salinity, Si decreased Na concentration and Na/K ratio in the shoot but not in the root. However, Si decreased net Na uptake and increased root Na accumulation content. Osmotic potential was increased in the shoot but decreased in the root by Si addition. Si decreased soluble sugar and proline concentrations in the shoot but increased soluble sugar and soluble protein concentrations in the root. Besides, Si promoted shoot transpiration rate and root morphological traits. Although both NaCl and PEG treatments upregulated aquaporin gene expression, Si addition maintained the expression of OsPIPs under NaCl and PEG treatments at same levels as control treatment. Furthermore, Si alleviated oxidative damages under both NaCl and PEG by regulating antioxidant enzyme activities. In summary, our results show that Si improves salt stress tolerance in rice by alleviating ionic toxicity and osmotic constraint in an organ-specific pattern. Si ameliorates ionic toxicity by decreasing Na uptake and increasing root Na reservation. Si alleviates osmotic constraint by regulating root morphological traits and root osmotic potential but not aquaporin gene expression for water uptake, and promoting transpiration force but not osmotic force in shoot for root-to-shoot water transport.Salinity stress severely inhibits the growth of plant via ionic toxicity and osmotic constraint. Exogenous silicon (Si) can alleviate salinity stress, but the mechanisms behind remain unclear. To investigate the role of Si in alleviating ionic and osmotic components of salinity, rice ( Silicon (Si) is the second most abundant element in soil and a widely recognized beneficial element in plant . The allWhen grown under salinity stress, plants suffer from two stress components: osmotic constraint and ionic toxicity . Excess Iso-osmotic growth conditions would be an effective method to discriminate the relative contribution of Si-induced ionic detoxification and osmotic stress alleviation to elucidate the underlying mechanisms. Upon dissecting the outcomes of researches aiming to distinguish the ionic effect from osmotic components on plant growth without Si addition, we know that a comparison between the effects of salinity and iso-osmotic conditions developed from non-penetrant chemicals  would be suitable. The ionic effect of salinity can be isolated by subtracting the osmotic effect from the total effects of salinity . In addiRice is one of the most salt-sensitive cereals and a major crop accounting for more food supply than any other crop. Besides, rice is a typical Si accumulator in which the Si transport system has been well-investigated. Thus, rice can benefit from Si addition at a relatively high level . In thisOryza sativa L. cv. Zhonghua11, ZH11) seeds were sterilized with 10% (v/v) H2O2 and then washed five times with deionized water. Seeds of rice were germinated on moist filter paper and then sown on sterilized quartz sand. After 5 days of growth, each 10 rice seedlings were transplanted to a plastic container filled with 3 L half-strength Kimura nutrient solution prepared as described by Rice ((w/v), respectively. For a better discrimination of the effects of Si in response to ionic and osmotic components of salt stress, 100 mM NaCl was used in this study  according to Uniform rice seedlings (28 days old) were used for treatment application. NaCl and PEG  were added with the final concentrations of 100 mM and 18% is study . The osmDry weight (DW) was determined after oven drying at 105°C for 1 h and at 80°C for 72 h. For root morphological traits analysis, fresh root samples were spread in a water layer immediately and scanned with a root scanner . Total root surface area, total root length, and root number (diameter > 0.5 mm) were analyzed using the software compatible to the root scanner.v/v) ethanol and measured at 665, 649, and 470 nm using an ultraviolet–visible (UV-vis) spectrophotometer . Chlorophyll concentration was calculated with the formula described by Pn) and transpiration rate (Tr) measurement with a portable photosynthesis system . Pn and Tr were calculated according to the leaf surface area in the test chamber.Chlorophyll was extracted with 95% (Relative water content (RWC) was measured following the procedure described by 3 and 1 ml H2O2 with a microwave digest system . The concentrations of Na and K were measured with a flame photometer .Dry shoot and root samples were ground into fine powder and digested in 5 ml HNOw/v) trichloroacetic acid solution. After centrifugation at 10,000 rpm for 10 min, the supernatant was mixed with 0.5% (w/v) thiobarbituric acid solution and incubated at 96°C for 20 min. Then, the reaction tubes were centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 600, 532, and 450 nm. The concentration of MDA was calculated according to For malondialdehyde (MDA) concentration measurement, rice samples were homogenized with 0.1% (Electrolyte leakage (EL) was measured with an electrical conductivity meter  according to w/v) polyvinyl pyrrolidone. The raw extract was centrifuged at 4°C for 20 min at 10,000 rpm, and the supernatant was used for the measurements of soluble protein concentration and antioxidant enzyme activity. Superoxide dismutase (SOD) activity was determined following the protocol of Fresh root and leaf samples were homogenized with 3 ml sodium phosphate buffer  containing 1 mM ethyl diamine tetraacetic acid and 2%  aqueous sulfosalicylic acid and incubated in boiling water for 10 min. Then, 1 ml extract was mixed with 1 ml glacial acetic acid and 1 ml ninhydrin solution. The mixture was incubated at 100°C for 1 h and cooled to room temperature; then, 5 ml toluene was added and mixed thoroughly. The absorbance of the upper phase was measured at 520 nm, and proline concentration was calculated with a standard curve. Soluble sugar was extracted from the powder of dry sample three times with 3 ml 80% (v/v) ethanol at 80°C, and the supernatant was collected after centrifugation at 10,000 rpm for 10 min each time. The extracts were mixed, and the total volume was adjusted to 10 ml. Soluble sugar concentration was measured with the anthrone-sulfuric acid method and calculated with a sucrose standard curve.The concentrations of osmolytes including proline, soluble sugar, and soluble protein were analyzed. Soluble protein was measured spectrophotometrically at 595 nm using the method described by OsPIP1;1, OsPIP1;2, OsPIP2;1, OsPIP2;2, OsPIP2;4, and OsPIP2;6) was determined by real-time quantitative PCR using TB Green premix Ex Taq  on a Lightcycler 480II system  following manufacturer’s instructions. OsActin1 was used as the internal reference. The sequences of primers used in this study are listed in For aquaporin gene expression analysis, rice seedlings were pretreated with or without 1.5 mM silicic acid for 3 days before NaCl or PEG was added. After 6 or 24 h of treatment, rice root samples were harvested and saved in liquid nitrogen. Total RNA was extracted using a plant RNA extraction kit  and treated with DNase I in the kit to remove genomic DNA. The isolated RNA was then converted to complementary DNA (cDNA) using a prime script RT reagent kit . The expression of aquaporin genes (including P < 0.05 [least significant difference (LSD)]. Plant growth data  were converted into a dissimilarity matrix using the Euclidean distances without standardization. For spatial ordinations, principal coordinate analysis (PCoA) was performed using the cmdscale function in “vegan” package of R (version 3.5.0)  and subjected to two-way analysis of variance (two-way ANOVA). Significant difference was determined with n 3.5.0) .Pn, Tr, chlorophyll concentration, and RWC in rice shoot (P < 0.05). However, there was no significant difference between root water status decline caused by NaCl or PEG  in rice shoot and root, while the alleviation effects on oxidative damages were more significant under NaCl than those under PEG. Moreover, the promotion effect of Si on RWC under NaCl was close to that under PEG in both the shoot and root  but not in rice root  induced no obvious effect on Na and K in rice seedling except a decline of plant total K accumulation content . Under Nice root . NaCl trice root . NaCl trice root . In addiice root . As for ice root . Si alsoice root . Moreoveice root  and alteice root .Exogenous Si significantly increased shoot sap osmotic potential but decreased root sap osmotic potential under both NaCl and PEG treatments . As for Generally, Si addition changed the activity of antioxidant enzyme and the regulation effects of Si differed with stress conditions . Si addiPIP gene family at 6 and 24 h after treatment were analyzed. The effects of Si on aquaporin gene expression varied among genes and stress conditions  is to subtract osmotic effect from total effects of salinity . HoweverIonic homeostasis is essential for the plant exposed to excess Na . Under sWhen subject to salinity stress, the shoot is more sensitive to Na than the root ; therefoMaintaining optimal water status by improving root water uptake and decreasing water loss is a major strategy for plant in the adaptation to salinity stress . PreviouOsPIPs expression, while Si decreased the expression of aquaporin genes under both NaCl and PEG treatments (PIPs have been reported in salt-stressed cucumber (OsPIPs could be an adaptation strategy to disrupted water status. In this study, Si addition promoted rice water status under NaCl and PEG treatments by increasing root–water contact area and osmotic force; consequently, the expression of OsPIPs remained unchanged relative to control treatment.In plant root, water uptake capacity mainly depends on root–water contact area, water uptake driving force, and root water permeability . Under weatments . Similarcucumber  and droucucumber . These rAfter uptake by the root, water is transported to the shoot driven by a combination of shoot osmotic force and transpiration force. Although the Si-improved transpiration rate may lead to more water loss, it can also drive more water transport from the root to the shoot. It is worth noting that the regulation effect of Si on osmotic potential and osmolytes in rice shoot was different from that in rice root. Si increased osmotic potential and decreased the concentrations of soluble sugar and proline in rice shoot . Given tIn addition, both ionic and osmotic stresses can cause membrane oxidative damage in plant , 2. OxidOur results indicate that Si improves salt stress tolerance in rice by alleviating ionic toxicity and osmotic constraint in an organ-specific pattern . Si amelAll datasets generated for this study are included in the article/GY and YL designed the study and wrote the manuscript. GY, XF, MP, CY, and ZX conducted the experiment and analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.