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Chymopapain. Chromatographic purification and immunological characterization.

Chymopapain (EC 3.4.22.6) was purified from commercially available spray-dried latex of papaya (Carica papaya) fruit by (NH4)2SO4 fractionation and fast protein chromatography on the Mono S cation-exchange column. Multiple forms of chymopapain separated chromatographically were shown to be immunologically identical. A major form was isolated and found to be homogeneous by several criteria, and fully active, and its N-terminal amino acid was identified as tyrosine. Latex from fresh unripe papaya fruit contained predominantly one form of chymopapain, and it is concluded that chymopapain is a single enzyme distinct from the other cysteine proteinases of C. papaya latex.

Amino Acids↗

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria.

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.

Animals↗

The application of a fermented food ingredient containing 'variacin', a novel antimicrobial produced by Kocuria varians, to control the growth of Bacillus cereus in chilled dairy products.

AIMS: The feasibility of applying variacin, a lantibiotic produced by Kocuria varians in the form of a spray-dried fermented ingredient to control the growth of psychrotrophic Bacillus cereus strains in chilled dairy foods, was evaluated. METHODS AND RESULTS: A range of chilled dairy food formulations modelling commercially-available products were fabricated, to which were added varying amounts of active ingredient. These were subsequently challenged with a B. cereus spore cocktail over a range of abuse temperatures. This work was validated by the inclusion of the fermented ingredient to commercial products. CONCLUSION: Results demonstrate the functionality of the bacteriocin at refrigeration abuse temperatures, and indicate the robust nature of the proteinaceous antimicrobial agent with regard to processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the applicability of fermented food ingredients containing naturally-occurring antimicrobials as additional hurdles in food preservation.

Actinomycetales↗

Effectiveness of muscimol-containing microparticles against pilocarpine-induced focal seizures.

PURPOSE: To investigate the efficacy of in situ lipid-protein-sugar particles (LPSPs) in mitigating the epileptogenic and histologic effects of intrahippocampal pilocarpine in rats. METHODS: LPSPs with and without muscimol were produced by spray-drying, sized by Coulter counter, and muscimol content determined by high-pressure liquid chromatography (HLPC). Particles, free muscimol or saline, were injected into the hippocampi of Sprague-Dawley rats before 40 mM pilocarpine, and seizure activity was scored. The trajectories of behavioral scores between groups were compared with two-way repeated measures analysis of variance. Animals were killed after 2 weeks. Brain sections were stained (Timm and thionin) and scored. RESULTS: LPSPs were 4 to 5 microm in diameter, and contained 0 or 2% (wt/wt) muscimol. In vitro, muscimol was released over a 5-day period. Intrahippocampal injections of normal saline and blank LPSPs did not deter seizure activity from pilocarpine. The rise of the trajectory in behavior scores in animals given LPSPs containing 5 microg muscimol was significantly slower than in those receiving saline, blank particles, or 5 microg of unencapsulated muscimol. There was less apparent neuronal injury and CA3 and supragranular mossy fiber sprouting in hippocampi of animals receiving muscimol-containing particles than in animals that did not receive muscimol. Hippocampi of animals that received 5 microg of encapsulated muscimol showed less supragranular sprouting than did those receiving 5 microg of unencapsulated muscimol, but showed no difference in cell loss or CA3 sprouting. CONCLUSIONS: Focally delivered biodegradable microparticles loaded with muscimol are effective in reducing seizure activity from pilocarpine in animals and mitigate the histologic effects.

Animals↗

Effects of the administration of Lactobacilli on body growth and on the metabolic profile in growing Maltese goat kids.

The aim of this study was to evaluate the effect of some lactobacilli on body growth and on the metabolic-nutritional status in growing goat kids. Twenty growing Maltese goat kids (10 Control and 10 Bios) were studied. The animals of the Bios group received a concentrate including 1 g x kg(-1) of SEB Bovino (spray-dried), Akron S.r.l., Italy, with non bacterial components: gum arabic, soybean meal, silicate alum of magnesium, and with bacterial components: 10(11) cfu kg(-1) each of Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus reuteri. Monthly, bio-metric and weight evaluations were carried out on each animal and individual blood samples were taken. The Bios group showed the highest body weight (Control 19 vs. Bios 23 kg P < 0.001), anamorphosis (Control 71 vs. Bios 78 P < 0.05) and body proportion (Control 35 vs. Bios 41 P < 0.001) indices; the lowest levels of Non Esterified Fatty Acids (Control 0.778 vs. Bios 0.403 mmol L(-1) P < 0.001), triglycerides (Control 0.21 vs. Bios 0.18 mmol L(-1) P < 0.05), urea (Control 8.83 vs. Bios 7.65 mmol L(-1) P < 0.05) and the highest levels of Alkaline Phosphatase (Control 270 vs. Bios 851 U L(-1) P < 0.01) and Creatine Kinase (Control 173 vs. Bios 285 U L(-1) P < 0.01). The results testify to the better metabolic activity of the Bios group which achieved, at the end of the trial (7 months old), about 99% of the morphological development of the adult, therefore an adequate structure for mating and going into production within the first year of life.

Animal Feed↗

Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies.

Escherichia coli is one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for economic losses due to mortality, morbidity, decreased growth rate, and cost of medication. The E. coli causing postweaning diarrhea mostly carry the F4 (K88) or the F18 adhesin. Recently, an increase in incidence of outbreaks of severe E. coli-associated diarrhea has been observed worldwide. The factors contributing to the increased number of outbreaks of this more severe form of E. coli-associated diarrhea are not yet fully understood. These could include the emergence of more virulent E. coli clones, such as the 0149:LT:STa:STb:EAST1:F4ac, or recent changes in the management of pigs. Development of multiple bacterial resistance to a wide range of commonly used antibiotics and a recent increase in the prevalence and severity of the postweaning syndromes will necessitate the use of alternative measures for their control. New vaccination strategies include the oral immunization of piglets with live avirulent E. coli strains carrying the fimbrial adhesins or oral administration of purified F4 (K88) fimbriae. Other approaches to control this disease include supplementation of the feed with egg yolk antibodies from chickens immunized with F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with zinc and/ or spray-dried plasma, dietary acidification, phage therapy, or the use of probiotics. To date, not a single strategy has proved to be totally effective and it is probable that the most successful approach on a particular farm will involve a combination of diet modification and other preventive measures.

Animals↗

Concurrent studies of the flow of digesta in the duodenum and of exocrine pancreatic secretion of calves. 3. Further studies on the addition of fat to skim milk and the use of non-milk proteins in milk-substitute diets.

1. The flow of digesta through the duodenum and the concurrent secretion of the pancreas were studied in four Friesian calves given four milk-substitute diets. The diets were: reconstituted, "mildly" pre-heated, spray-dried skim-milk powder with (SKF) or without (SK) margarine fat or with 5o percent of the skim-milk powder in diet SKF replaced by soya-bean flour (ASKF) or fish-protein concentrate (BSKF), together with dried whey. The diets were given ad lib. twice daily from 13 to 37 d of age, each diet being given for 6 consecutive days. Collections of duodenal digesta and pancreatic secretions, from cannulas, were made for 12 h after feeding the 6th and 12th meals ("experimental" meals) for each diet. 2. The diets fed as "experimental" meals contained polyethylene glycol (PEG) as a fluid (whey) marker and goat's milk containing (3H)lysine as a marker for total protein; beta-carotene was added as a lipid marker to the three diets containing margarine fat. 3. Over the 12 h postprandial period, the patterns of duodenal digesta flow and secretion of pancreatic fluid did not differ markedly between the four diets. The abomasal outflow of both nitrogen and lipid in a 12 h postprandial period was related to their intakes from the "penultimate" (5th and 11th) meals for diets SKF and SK BUT TO THEIR INTAKES AT THE "EXPERIMENTAL MEALS" FOR DIETS ASKF and BSKF. Secretion of pancreatic enzyme activity was highest during the 1st hour after feeding but the main outflow from the abomasum of total N and lipid occurred 5-10 h after feeding. 4. The time required for all the whey marker (PEG) to pass through the duodenum was similar for diets SKF and SK, but only 53 and 42 percent respectively of the ingested protein marker passed through the duodenum in the 12 h after feeding. More acid appeared to be secreted by the abomasum when diet SK was given; also less undigested protein passed out of the abomasum after giving this diet. It is concluded that the physical absence of fat globules in the abomasal clot increases the degree of proteolysis. 5. The secretions of pancreatic fluid and pancreatic enzyme activity were all markedly lower for diet SK than for diet SKF. 6. With diets containing non-milk proteins (ASKF and BSKF), abomasal proteolysis was less efficient and the ingested protein passed out of the abomasum more rapidly than for diet SKF. There was no difference in the rate of abomasal outflow of the whey fluids between diets SKF, ASKF and BSKF. 7. In comparison with diet SKF, diets ASKF and BSKF tended to induce less pancreatic enzyme secretion over a 12 h postprandial period, with the exception of lipase. 8. There appeared to be no direct relationship between the quantities of any of the pancreatic enzymes secreted during a postprandial period and either the concurrent flow of duodenal digesta or the total quantities of dietary constituents passing through the duodenum.

Animal Nutritional Physiological Phenomena↗

Concurrent studies of the flow of digesta in the duodenum and of exocrine pancreatic secretion of calves. 7. Influence of milk substitutes on abomasal lipolysis and biliary secretion.

1. The abomasal hydrolysis of lipids and the flow of endogenous (biliary) lipids was studied in two Friesian calves given four milk-substitute diets, by sampling the duodenal digesta. The diets were: reconstituted, mildly preheated, spray-dried skim-milk powder with (SKF) or without (SK) margarine fat or with 500 g/kg skim-milk powder in diet SKF replaced by soy-beran flour (ASKF) or fish-protein concentrate (BSKF) together with dried whey. The diets were given ad lib. twice daily from 13 to 37 d of age, each diet being given for six consecutive days. Collections of duodenal digesta from the re-entrant cannula situated caudal to the bile duct were made for 12 h after feeding the 6th and 12th meals for each diet. Samples from one collection only were subjected to detailed analysis of the lipid classes. 2. The inclusion of non-milk protein (ASKF and BSKF) not only increased the rate of passage of lipid through the abomasum but also the proportion of the lipid present as triglyceride particularly, in the first 2 h after feeding. 3. In a 12 h period, 2.3-6.3 g 'polar' lipids (mainly biliary phospholipids) were estimated to have been secreted. The rate of flow was high during the first hour after feeding and constant thereafter. The quantity of 'polar' lipid was not related to the type of milk fed or the duodenal flow of lipid. 4. When diet SK was fed, the small amounts of lipid present were extensively hydrolysed so that free fatty acids represented 700 g/kg lipid of dietary origin passing through the duodenum. When margarine fat was included in the diets (SKF, ASKF and BSKF), the free fatty acids represented only 210 g/kg lipid of dietary origin. 5. The quantities of lipid and nitrogen passing through the duodenum were poorly related to the quantities ingested at the beginning of the 12 h experimental period but were closely related to each other.

Abomasum↗

A comparison of iron absorption in adults and infants consuming identical infant formulas.

Fe absorption was estimated in adults and infants from the erythrocyte incorporation of Fe isotopes added to infant formula. Fe absorption was measured in adults using radioisotopes, and in infants with a stable-isotope technique. In adults, the geometric mean Fe absorption from a ready-to-feed soya formula with its native phytic acid content was 2.4%. This increased to 6.0% (P < 0.05) after almost complete dephytinization. In infants, mean Fe absorption values were 3.9 and 8.7% respectively from the same products (P < 0.05). In adults, mean Fe absorption from a spray-dried soya formula containing 110 mg ascorbic acid/l was 4.1%, increasing to 5.3% (P < 0.05) when ascorbic acid was doubled to 220 mg/l. In infants, mean Fe absorption values were 5.7 and 9.5% (P < 0.05) from the same products. Mean Fe absorption from a milk-based formula was 6.5% in adults compared with 6.7% in infants. All meals in the adult and infant studies were fed using an identical meal size of 217 g. Increasing the meal size threefold in adults did not change fractional Fe absorption. Mean Fe absorption values for each meal were lower in adults than in infants but the relative inhibitory effect of phytic acid and the enhancing effect of ascorbic acid were similar. We conclude that Fe absorption studies in adults can be used to assess the influence of enhancers and inhibitors of Fe absorption in infant formulas fed to infants. Further studies, however, are required to extend these findings to weaning foods and complete meals.

Adult↗

Effects of the whole seed and a protein isolate of faba bean (Vicia faba) on the cholesterol metabolism of hypercholesterolaemic rats.

The aim of the present work was to analyse the hypocholesterolaemic efficiency of a Vicia faba-protein isolate in relation to the intact legume. In addition, the mechanisms underlying the effects of this isolate were investigated. Hypercholesterolaemic rats were divided into three groups and fed high-fat diets rich in cholesterol-containing casein, whole seeds of Vicia faba or the protein isolate of faba beans as protein source, for 2 weeks ad libitum. The protein isolate was prepared by isoelectric precipitation and spray dried. Analyses of serum, liver and faeces, as well as of the activity of hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, were assessed by enzymatic methods. The rats fed on Vicia faba diets showed significantly lower body weights and energy intakes than rats fed on casein diets. The whole-seed diet induced a significant reduction in plasma triacylglycerol. Feeding rats on diets containing faba bean seeds, or the protein isolate, induced a significant decrease in plasma (LDL+VLDL)-cholesterol but not in HDL-cholesterol. Hepatic cholesterol and triacylglycerol were also reduced. The hypocholesterolaemic effects of Vicia faba were not the result of a reduction in cholesterol synthesis as assessed from HMG-CoA reductase activity, but the result of an increase in steroid faecal excretion. The faba bean-protein isolate obtained under our experimental conditions was useful in improving the metabolic alterations induced by feeding with a hypercholesterolaemic diet compared with casein. The effectiveness of the whole seeds was higher than that of the protein isolate.

Analysis of Variance↗

Effect of a buccal bioadhesive nystatin tablet on the lifetime of a Provox silicone tracheoesophageal voice prosthesis.

CONCLUSION: Daily application of a buccal bioadhesive slow-release nystatin tablet (100,000 IU per tablet) significantly increased the voice prosthesis lifetime in laryngectomized patients compared to conventional local cleaning of the prosthesis with an antimicrobial agent on a brush. OBJECTIVE: To investigate the effect of a buccal bioadhesive nystatin tablet on the lifetime of a Provox tracheoesophageal voice prosthesis in post-laryngectomy patients. MATERIAL AND METHODS: A buccal bioadhesive tablet, based on a spray-dried Amioca/Carbopol 974P mixture containing 10% (w/w) Carbopol 974P, was loaded with 100,000 IU of nystatin. Patients were included in the study when replacement of their voice prosthesis was required and were divided into three groups. Conventional daily local cleaning of the voice prosthesis by means of an oral nystatin suspension on a brush (Group 1; n = 7) was compared with application of one nystatin buccal bioadhesive tablet per day, after breakfast, on the gingiva above the upper canine (Group 2; n = 7). The control group (n = 5) used no antimicrobial agents. The lifetime of the prosthesis was followed and expressed in days. RESULTS: The lifetime of the voice prosthesis was significantly increased in Group 2 compared to Group 1 (p < 0.05; paired t-test), indicating that sustained release of nystatin in the oral cavity, by means of erosion of the tablet over a period of approximately = 8 h, is more effective at preventing microbial colonization of the prosthesis than local cleaning.

Acrylic Resins↗

A successful attempt to raise goat kids free of infection with caprine arthritis encephalitis virus in an endemically infected goat herd.

Goat kids from a herd endemically infected with caprine arthritis encephalitis (CAE) virus were raised according to 3 methods. One group of ten goat kids was removed from infected does at birth before suckling or licking by the doe could occur (snatch birth technique). Kids were fed on goat colostrum, which had been heated to 57 degrees C for ten minutes and then held in a thermos flask for one hour. Subsequently the kids were fed reconstituted spray dried cows' milk powder. They were raised apart from infected goats with separation maintained by a wire fence. Contact occurred across-the-fence. Passively acquired serum antibody to CAE virus was detected in some kids at two to three months of age. Nine of the ten goats were negative for serum antibody to CAE virus when tested at 5-6, 9 and 12 months of age. One goat was positive at three and nine months of age but was negative when tested at 12 months of age. A second group of four kids was removed at birth and fed heat-treated goat colostrum, followed by milk from CAE virus-infected does. All four kids became infected with CAE virus; they developed serum antibody to CAE virus between 5-6 and 9 months of age. A third group of two kids was not removed from their infected dams. Both kids were infected at 5-6 and 9 months of age.

Journal Article↗

Dietary lipid level and the availability of beta-carotene of Dunaliella-bardawil in rats.

High plasma and tissue levels of beta-carotene have been implicated to be inversely related to the incidence of some types of cancer. Previous studies indicated that the availability of the natural beta-carotene isomer mixture of Dunaliella bardawil was severalfold higher than that of synthetic all-trans beta-carotene. This study illustrated that this higher availability, as expressed by hepatic and plasma beta-carotene, can be further enhanced by increased dietary lipid level. For 12 days, weanling male and female rats were fed diets containing either 0.1% all-trans beta-carotene or spray-dried Dunaliella bardawil having an equal level of the beta-carotene isomer mixture. The diets contained 5 or 15% soybean oil. The rise in dietary fat resulted in a marked increase in plasma and liver beta-carotene, with the Dunaliella isomer mixture being more effective. Higher levels were found in female than in male animals. These findings may be relevant when a high dose of beta-carotene is considered as a potential anticancer agent.

Analysis of Variance↗

The effect of dietary fermented milk products and lactic acid bacteria on the initiation and promotion stages of mammary carcinogenesis.

The effects of spray-dried yogurt powder product (YPP), bifidobacteria, and Lactobacillus acidophilus were studied during the initiation and promotion phases of carcinogenesis using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse mammary carcinogenesis model. In two separate studies, Sencar mice were fed a diet consisting of 86%, 43%, or 0% YPP or 0% YPP, but with added cultures of bifidobacteria or L. acidophilus. When the animals were 55-63 days old, DMBA was administered by intragastric gavage at 1 mg/mouse and continued once a week for six weeks. During the initiation study, the test diets were fed for four weeks before and during DMBA administration. One week after the final DMBA treatment, all animals were switched to a basal diet based on the AIN-76 formulation. For the promotion study, the diets were introduced one week after the final dose of DMBA and fed for the remainder of the study. Palpable tumor development was monitored weekly throughout the studies. For the initiation study, mice fed 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus had a histologically verified mammary tumor incidence of 15%, 35%, 19%, 30%, and 20%, respectively. The histologically verified tumor incidence for the promotion study was 48%, 58%, 36%, 59%, and 43% in the mice fed diets consisting of 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus, respectively. The data indicate that neither the initiation nor the promotion phase of carcinogenesis is significantly affected by diets composed of 86% YPP, 43% YPP, 0% YPP, or 0% YPP supplemented with bifidobacteria or L. acidophilus.

9,10-Dimethyl-1,2-benzanthracene↗

Gum arabic (Acacia senegal): unambiguous identification by 13C-NMR spectroscopy as an adjunct to the Revised JECFA Specification, and the application of 13C-NMR spectra for regulatory/legislative purposes.

The JECFA Specification for gum arabic was revised in 1990 to reflect more closely the specification of the Test Article used in evaluations that led to its classification 'ADI not specified' in 1982/83. Some producers and traders have objected to the Revised Specification; in contrast, consumer-protection groups consider that it remains too lax to provide the degree of safety assurance expected. This paper presents analytical data that confirm the mean values previously established for nitrogen and the specific rotation of bulk commercial gum arabic from Acacia senegal. The data also establish that natural gum arabic imported into the USA and Europe in 1989/91 met the Revised Specification, but that a disturbingly high proportion of spray-dried, processed gums sold as 'gum arabic' did not. NMR spectroscopy has (a) indicated that some such samples are based on non-permitted gums and (b) confirmed that the 1983 Test Article represents not only typical 1990/91 shipments but also a wide range of reference gum arabic samples from other reputable sources. Details of a representative 13C-NMR spectrum, derived by averaging the relative intensities for the characteristic resonances of 35 gum arabic samples, are given for future regulatory/legislative purposes. Some limitations of the Revised Specification and its susceptibility to commercial exploitation are discussed.

Carbon Isotopes↗

Ciprofloxacin-loaded bovine serum albumin microspheres: preparation and drug-release in vitro.

Ciprofloxacin albumin microspheres were prepared by the spray drying technique, with bovine serum albumin as the natural biodegradable wall material. The spherical microspheres, flowed well, were organic solvent free and in the size range 1-5 microm. The drug release from the microspheres could be retarded by further thermal denaturation. The sustained-release microspheres were suitable for dry powder inhaled lung drug delivery systems.

Animals↗

Protein immobilization in crosslinked alginate microparticles.

Oral administration of peptide and protein drugs requires their protection from the acidic and enzymatic degradation in the gastro-intestinal environment and their targeting to the absorption zone. For this purpose, an alginate microsystem, as a carrier of bovine serum albumin (BSA), as a model protein, was developed using a spray-drying technique. A hardening process with Ca2+ and chitosan (CS) provided a system with resistance to the gastro-intestinal barriers and of appropriate size for targeting to the Peyer's patches. The present work aims to evaluate the effects of the ratio of sodium alginate (Na-A) and BSA as well as the pH of the crosslinking medium on the microsystem properties. Microparticle morphological and dimensional characteristics did not change significantly with the formulation variables. BSA loading at a pH value less than the protein isoelectric point (pI) was higher than that at a pH similar to the pI owing to an electrostatic interaction between the charged protein and the polyanionic alginate. The maximum encapsulation efficiency was obtained at the highest Na-A/BSA ratio. Protein release in a simulated gastro-intestinal fluid was not affected by the preparative variables, but was controlled by the pH-dependent nature of the polymer material. Polyacrylamide gel electrophoresis (PAGE) demonstrated the stability of the protein to both the preparative conditions and the gastro-intestinal pH values.

Alginates↗