Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) protein

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can influence the digestion of plant protein in the rumen. We compared the effects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens ) was incubated with fresh rumen fluid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantified by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and imaging densitometry. To deduce the effects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts differed markedly in their effects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extract from L pedunculatus was more effective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg ⁻¹ total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which affects the activity of the CT needs to be considered when assessing the effects of CT on protein metabolism in ruminants. © 1999 Society of Chemical Industry     

Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid

This experiment aims to determine whether polyphenol oxidase (PPO) can reduce the extent of lipolysis and the consequent polyunsaturated fatty acid loss through microbial biohydrogenation in red clover when incubated in the presence of rumen fluid. PPO is involved in the browning reaction of red clover leaves when cut or crushed and exposed to air. It starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins and have been shown to reduce proteolysis and lipolysis in silo. Two lines of red clover (cv. Milvus), a genotypic mutant with reduced PPO activity (L) and the wild type (H) with a high level of PPO activity, were cut 3 cm above soil level, crushed and cut into 1 cm strips before being loaded into incubation bottles. These were then incubated in anaerobic buffer at 39 °C in either the absence (?) or the presence (+) of rumen microorganisms. The incubations were then compared over a 24 h time course in terms of lipolytic activity. Characterization of the tissues showed PPO activities of 25.3 and 5.13 U g^?1 fresh weight for H and L, respectively. Lipolysis, measured as the proportional decline in the membrane lipid, was reduced (P < 0.001) with increasing PPO activity in both the presence (+) and absence (?) of rumen microorganisms. However, values were significantly higher (P < 0.001) in the presence of rumen microorganisms, with values after the 24 h incubation of 0.28, 0.42, 0.72 and 0.82 for H?, L?, H+ and L+, respectively. Biohydrogenation of C18:2 and C18:3 polyunsaturated fatty acids were significantly lower in the H+ treatment than the L+ treatment, with mean values after 24 h incubation of 53% and 57% (P < 0.05) for C18:2 and 65% and 74% (P < 0.01) for C18:3, respectively. Changes that occurred in the lipid fractions (membrane lipid, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce polyunsaturated fatty acid loses in the rumen. Copyright © 2007 Society of Chemical Industry     

Evaluation of the effect of formic acid and level of formaldehyde application before ensiling on silage protein degradability

The effect of the application, before ensiling, of formic acid alone, and together with increasing levels of formaldehyde, on the degradability of the protein of ryegrass and red clover silages has been assessed on the basis of nitrogen solubility in mineral buffer; susceptibility of N to degradation during in-vitro incubation with either rumen microorganisms, acid pepsin or neutral protease; and N disappearance when the silages were incubated in situ in Dacron bags in the rumen of sheep receiving dried grass. The relative effects of the additives were generally consistent for both crops and with all procedures: formic acid either had no effect or reduced degradability by only a small amount, whereas a mixture of formic acid and formaldehyde was more effective than formic acid alone in protecting protein from degradation, and degradability decreased in a curvilinear manner with increasing levels of formaldehyde application. Absolute values for protein degradability based on buffer solubility and in-vitro degradation by rumen microorganisms were very similar but lower than those based on digestion with proteolytic enzymes which in turn were lower than those obtained with the rumen in situ procedure. Buffer solubility and in-vitro incubation with rumen microorganisms also showed much bigger differences between the formic acid-treated and the formaldehyde-treated silages than the other methods.     

Nitrogen metabolism in the rumen

Protein metabolism in the rumen is the result of metabolic activity of ruminal microorganisms. The structure of the protein is a key factor in determining its susceptibility to microbial proteases and, thus, its degradability. Ruminal protein degradation is affected by pH and the predominant species of microbial population. Ruminal proteolytic activity decreases as pH decreases with high-forage dairy cattle-type rations, but not in high-concentrate beef-type rations. Accumulation of amino acid (AA) N after feeding suggests that AA uptake by rumen microorganisms could be the limiting factor of protein degradation in the rumen. In addition, there are several AA, such as Phe, Leu, and Ile, that are synthesized by rumen microorganisms with greater difficulty than other AA. The most common assessment of efficiency of microbial protein synthesis (EMPS) is determination of grams of microbial N per unit of rumen available energy, typically expressed as true organic matter or carbohydrates fermented. However, EMPS is unable to estimate the efficiency at which bacteria capture available N in the rumen. An alternative and complementary measure of microbial protein synthesis is the efficiency of N use (ENU). In contrast to EMPS, ENU is a good measurement for describing efficiency of N capture by ruminal microbes. Using EMPS and ENU, it was concluded that optimum bacterial growth in the rumen occurs when EMPS is 29 g of bacterial N/kg of fermented organic matter, and ENU is 69%, implying that bacteria would require about 1.31 x rumen-available N per unit of bacterial N. Because the distribution of N within bacterial cells changes with rate of fermentation, AA N, rather than total bacterial N should be used to express microbial protein synthesis.     

Studies on rumen metabolism. IX. Contribution of plant lipases to the release of free fatty acids in the rumen

The role of plant lipases in the hydrolysis of dietary lipids in the rumen of pasture-fed ruminants has been investigated by means of in vitro and in vivo experiments with rumen contents. Lipases present in the leaves of numerous pasture plants remained highly active for at least 5 h in the presence of metabolising rumen microorganisms, leading to the hydrolysis of triglyceride. Parallel experiments showed that the lipase activity of actively metabolising micro-organisms in rumen fluid was very low. A slight increase in lipolytic activity attributable to micro-organisms occurred after incubations of about 4 h with plant extract when the metabolic activity of the microbial population had passed its peak. In vivo experiments showed that the lipolytic activity of rumen fluid, obtained 0.5 to 5.0 h after feeding fresh grass, was about twice that of rumen fluid obtained after overnight fasting. Paired feeding experiments showed that lipase activity was higher in rumen fluid obtained from a pasture-fed, than from a hay-fed animal. Nevertheless, there was appreciable activity in the rumen contents obtained from the hay-fed twin, which is consistent with the presence of lipase activity in the extracts of dried grass. Multiple forms of plant esterases and phosphatases were present in the soluble fraction of rumen fluid several hours after feeding. It is concluded that the rapid release of free fatty acids which follows the ingestion of pasture is due mainly to the activity of plant enzymes and that rumen microorganisms have a subsidary role in hydrolysing ingested lipid.     

Ruminal micro‐organisms do not adapt to increase utilization of poly‐phenol oxidase protected red clover protein and glycerol‐based lipid

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants with subsequent increases in the efficiency of N utilization and the level of beneficial polyunsaturated fatty acids in their products (meat and milk). It has also been reported that red clover feeding alters the rumen microbial population compared to grass feeding. This study investigated whether the observed shifts in the microbial population of the rumen when ruminants are fed red clover silage (RC) as opposed to grass silage (G) represented an adaptation by the micro‐organisms to increase the utilization of PPO‐protected protein and glycerol‐based lipid. RESULTS: The experiment consisted of two periods where ruminally fistulated dairy cows were offered either RC or G for 2 weeks, followed by collection of rumen fluid, which was then used in in vitro incubations to investigate lipolysis and proteolysis over time in plant material derived from red clover plants with either wild type PPO expression (PPO+) or PPO expression reduced to undetectable levels by gene silencing (PPO?). Proteolysis and lipolysis (P < 0.05) were lower after 24 h of incubation in the PPO+ treatment than the PPO? treatment irrespective of rumen fluid. Biohydrogenation of C18 polyunsaturated fatty acids was also lower on the PPO+ treatment than the PPO? treatment, with no effect of rumen fluid. CONCLUSION: These results suggest that microbial changes to red clover feeding did not result in an increased ability of the micro‐organisms in the present study to utilize either PPO‐protected protein or glycerol‐based lipid. Copyright © 2008 Society of Chemical Industry     

Characterization of proteolysis during the ripening of semi-dry fermented sausages

The respective contribution of indigenous enzymes and enzymes from starter bacteria to proteolysis in fermented sausages were determined by comparing the proteolytic changes occurring in sausages resulting from the presence of a proteolytic strain of Staphylococcus carnosus, i.e. S. carnosus MC 1 to the proteolytic changes occurring in control sausages containing glucono-δ-lactone (GDL) and an antibiotic mixture. Proteolysis was quantified by assaying for non-protein nitrogen (NPN) and free amino acids. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reversed phase high performance liquid chromatography (RP-HPLC) were used to qualitatively assess the proteolytic changes in the sarcoplasmic and myofibrillar proteins as ripening progressed. The concentration of NPN and free amino acids increased in both sausages initially, but subsequently decreased towards the end of ripening in sausages inoculated with the starter culture. SDS-PAGE showed a similar pattern of proteolysis of sarcoplasmic proteins in both sausages, while of the two sausage types; the S. carnosus MC 1 inoculated sausages exhibited the most intense degradation of myofibrillar proteins, especially myosin and actin. RP-HPLC profiles of 2% trichloroacetic acid (TCA)-soluble peptides for the two sausage types were similar, with the production of numerous hydrophilic peptides. N-Terminal amino acid sequence analysis and sequence homology with proteins of known primary structure showed that six of the TCA-soluble peptides were released from the sarcoplasmic (myoglobin and creatine kinase) and myofibrillar (troponin-I, troponin-T and myosin light chain-2) proteins. In addition, the initial degradation of sarcoplasmic proteins was due to the activity of indigenous proteinases, while both indigenous and bacterial enzymes contributed to the initial degradation of myofibrillar proteins. Furthermore, indigenous enzymes were responsible for the release of TCA-soluble peptides, which, were further hydrolysed by bacterial enzymes.     

Antimicrobial peptides generated from milk proteins: a survey and prospects for application in the food industry. A review

Milk proteins constitute a natural reservoir of bioactive peptides with physiological and/or antimicrobial properties, the release of which requires hydrolysis of the precursor molecules by digestive proteases or by fermentation with proteolytic micro‐organisms. Depending on the digestive or microbial proteases used, an array of bioactive peptides would be released either from caseins or whey proteins, but only a small part of these peptides has so far been identified and characterised with respect to their antimicrobial activity. The antimicrobial peptides known thus far have proven to be potent inhibitors to the growth of a wide range of undesirable micro‐organisms of health or spoilage significance. Nevertheless, previous research work has largely been oriented towards their possible application in medicine, which has hindered their high potential as food‐grade biopreservatives and/or as supplements in functional foods. This review attempts to study the literature pertaining to antimicrobial peptides derived from major milk proteins (caseins, α‐lactalbumin and β‐lactoglobulin) upon hydrolysis either by digestive proteases or by fermentation with proteolytic lactic acid bacteria. Their possible application in the food industry and their mechanism of action will also be discussed. Reference antimicrobial peptides produced by living micro‐organisms as innate immune defence components against microbial infections will occasionally be invoked for comparison purposes.     

Silage fermentation of fish or fish waste products with lactic acid bacteria

Silage fermentation of minced fish and fish offal after inoculation with cereals prefermented with Pediococcus acidilactici and Lactobacillus plantarum initiates a rapid fall in pH to below 4.5 within 30 h, and the content of competing gram-negative fermenters and fish pathogens, such as Vibrio anguillarum and Aeromonas salmonicida, is eliminated. The addition of 0.1% sorbic acid inhibits the growth of yeasts during the initial fermentation period and during storage but does not affect the lactic acid fermentation. Degradation of nitrogen components proceeds during storage and is manifested as an increase in volatile basic nitrogen compounds, amino acids and peptides. These substances increase the pH and/or force the bacteria to produce more acid. The rate of production of these basic substances is mainly temperature-dependent and cannot be attributed to microbial activity. The proteolytic activity is mainly caused by tissue proteases (e.g., cathepsins) and, to a lower degree, by gut proteases. It is doubtful whether fish fermentation can be utilised on an industrial scale without improving the technique. Such improvements consist of the selection and use of psychotrophic lactic acid fermenters and commercially acceptable inhibitors of proteolytic activity and yeast growth.     

Effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and nutrient utilization in dairy does

The objective of this study was to evaluate effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and on feed intake and nutrient utilization in dairy does. Batch cultures of mixed rumen microorganisms were used to study effects of different concentrations of fumarate on fermentation with various N sources (ammonia as ammonium bicarbonate, casein amino acids, casein peptides, gelatin peptides) and feeds (bermudagrass hay, mixed diet of 60% bermudagrass hay plus 40% concentrate) for 6 and 24 h, respectively. Substrates were grouped into pairs for separate incubations. Monosodium fumarate was added to incubation tubes to achieve final concentrations of 0, 5, and 10 mM fumarate. More ammonia accumulated at the end of incubation with added ammonium bicarbonate. Ammonia concentration was higher for peptide compared with amino acid incubation, and for casein peptide compared with gelatin peptide. Addition of fumarate linearly decreased ammonia for all N sources and for feed substrates. For all substrate types, fumarate treatment increased acetate, propionate, and total volatile fatty acids (VFA), decreased acetate to propionate ratio, and tended to reduce branched-chain VFA. Digestion of feed neutral detergent fiber (NDF) by rumen microorganisms was improved by fumarate along with elevated endoglucanase and xylanase activities. In an animal metabolism experiment, 8 dairy does (4 per treatment) were used in a completely randomized design for 21 d. Does were fed a hay plus concentrate diet without (control) or with fumarate (6 g/head per day) supplementation to determine feed intake, whole-tract nutrient digestibility, and N utilization. Fumarate treatment did not affect weight change or feed intake but increased whole-tract digestion of gross energy, crude protein, and cellulose. Digested N was increased by fumarate supplementation; however, N retention was unaffected. Plasma glucose concentration was elevated with fumarate but urea N concentration remained unchanged. Fumarate addition had significant effects on rumen microbial fermentation by decreasing ammonia and branched-chain VFA, and by increasing acetate and propionate, and NDF digestion. These effects were reflected in the improvement in whole-tract gross energy, crude protein, and cellulose digestion and elevated plasma glucose concentration when dairy does were supplemented with fumarate.     

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hard-cooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 ??m). The substrates were incubated at 34 °C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37 °C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.     

Recent advances in rumen microbial ecology and metabolism: potential impact on nutrient output

Feedstuffs consumed by ruminants are all initially exposed to fermentative activity in the rumen prior to gastric and intestinal digestion. The extent and type of transformation of feedstuffs thus determines the productive performance of the host. Research on rumen microbial ecology and metabolism is essentially a study of the interactions between the host, microorganisms present, substrates available, and end products of digestion. Furthermore, the interactions of the normal microbial flora with the host can be manipulated to improve the efficiency of nutrient utilization in ruminant animals. Three important areas of ruminal fermentation will be reviewed, N metabolism, fiber degradation, and biotransformation of toxic compounds. The extent of protein degradation and the rate of uptake of resultant peptides and ammonia are extremely important factors in determining the efficiency of N utilization by rumen bacteria and, therefore, the relative amounts of microbial or bypass protein available to the host. Strategies aimed at identifying and characterizing rate-limiting enzymes of cellulolytic bacteria are essential in elucidating mechanisms involved in ruminal fiber degradation. Results obtained with ruminococci will be described. The detoxification of phytotoxins by passage through the gastrointestinal tract of ruminants is a process deserving special attention and several examples will be presented. Opportunities for manipulation of rumen fermentation are good. However, successful manipulation and full exploitation depend on a through understanding of the mechanisms involved.     

Comparison of grass and legume silages for milk production. 2. In vivo and in sacco evaluations of rumen function

Two experiments were conducted to investigate the basis for higher voluntary intakes and increased alpha-linolenic acid content in milk from cows offered clover silages. Six cows with rumen and duodenal cannulae were used in a four-period changeover-design experiment. Cows received 8 kg/d of dairy concentrate and had ad libitum access to one of six silage treatments: grass, red clover, white clover, alfalfa, and 50/50 (dry matter basis) mixtures of grass with red clover or white clover. The rumen fermentability of grass, red clover, white clover, and grass/red clover silages was also evaluated in a nylon bag study. Legume silages led to increased dry matter intake and milk production in comparison with grass silage. There was no significant effect of legume silages on rumen pH and volatile fatty acid concentrations, but a significant increase in rumen ammonia concentration with the legume silages, reflecting their higher protein content. The inclusion of white clover or alfalfa silage, but not red clover silage, in diets led to an increase in molar proportions of isobutyric, iso-valeric, and n-valeric acids in comparison with diets based on grass silage. Rumen fill was significantly lower, and rumen passage rates were significantly higher for cows offered alfalfa or white clover silages. However, the markedly different particle size distribution of rumen contents with these feeds suggests very different mechanisms for the high intake characteristics: high rates of particle breakdown and passage with alfalfa, and high rates of fermentation and passage with white clover. Microbial energetic efficiency (grams microbial N per kilogram organic matter apparently digested in the rumen) was highest for cows offered alfalfa silage, intermediate for clover silage, and lowest for cows offered grass silage. These differences reflect the higher rumen outflow rates for legume silages in comparison with grass silage. However, the effect of these differences on N-use efficiency (feed to milk) was probably quite small in comparison with effects of N intake. Although the biohydrogenation of alpha-linolenic acid was still high for red clover silage (86.1% compared with 94.3% for grass silage), there was a 240% increase in the proportion of alpha-linolenic acid passing through the rumen. This explains the increased recovery of alpha-linolenic acid from feed into milk with diets based on red clover silage.     

The buffering constituents of herbage and of silage

The buffering capacity values of several herbage species and of silage made from this herbage, and the contributions of plant constituents to this buffering, between pH 4 and 6, were determined, and found to vary with the herbage species; values in the silages were normally two to three times greater than those in the plant materials. The anion fraction of the plant materials accounted for 68–80% of the total buffering capacity, and for 73–88% in the silages. Buffering caused by plant proteins was estimated to be 10–20% of the total buffering capacity. The buffering capacity of wilted red clover (Trifolium pratense) was 18% lower than that of fresh red clover, and the total organic acid content of wilted clover was also lower than that in fresh clover. The organic acids were responsible for most of the buffering effect in herbages and silages. In Italian ryegrass (Lolium multiflorum), the main buffers were malate and citrate, but, in red clover, glycerate and malate were the main buffers. The clovers studied contained a high level of glycerate (4% of dry matter). During ensilage, malate, citrate and glycerate were extensively broken down. The increased buffering capacity during ensilage was caused mainly by the formation of lactates and acetates.     

Chicken pancreatic enzymes for clinical use: Autoactivation of the proteolytic zymogens

The potential of chicken pancreatic enzymes for use as digestive aids was investigated. Frozen chicken pancreas was pulped and the enzymes were extracted at pH 8.0 and 6.0. The extracts were incubated for 165 h at either 0 or 5°C in order to induce autoactivation of the proteases. Extracts at pH 8.0 showed a faster activation rate of trypsin up to 85 h compared with pH 60 extracts, and pH 60 extracts showed a slightly higher trypsin activity upon termination of the incubation period. Higher initial chymotrypsin activity and total proteolytic activity were observed in pH 8.0 than in pH 6.0 extracts. Chymotryptic and total proteolytic activity showed a similar pattern, reaching maximal levels after 42 h incubation. Lipase and amylase activity declined with the increase in proteolytic activity.     

Production of proteases by psychrotrophic microorganisms.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.     

In-vitro rates of rumen proteolysis of ribulose-1,5-bisphosphate carboxylase (rubisco) from lucerne leaves,and of ovalbumin,vicilin and sunflower albumin 8 storage proteins

Laboratory systems were developed, based upon in-vitro incubations with rumen fluid, to examine the rate of proteolysis (ie degradation) of sunflower albumin 8 (SFA 8; 24% sulphur amino acids; SAA) from sunflower seeds, ovalbumin (6% SAA) from chicken egg white, ribulose-1,5-bisphosphate carboxylase (Rubisco; 3% SAA) from lucerne leaves, and vicilin (0% SAA) from pea seeds. After fractionation by SDS-PAGE, proteins were analysed by either Western blotting, using specific antibodies (SFA 8, vicilin and ovalbumin) or by Coo-massie blue staining (Rubisco). Proteolysis of the large subunit of Rubisco occurred very quickly and as two components, with half-lives of 11.6 and 1.5 h. The small subunit of Rubisco was more resistant to degradation, with a half-life of 17.3 h. Vicilin was degraded extremely rapidly (half-life 0.16 h). SFA 8 and ovalbumin both showed resistance to degradation, but by two different mechanisms. Ovalbumin was not degraded at all during the initial 16 h of incubation, but then degraded with a half-life of 8.7 h. SFA 8 (mol wt 12 100) disappeared very rapidly, with a half-life of 3.0 h. The degradation of SFA 8 was associated with the appearance of a polypeptide (mol wt 8000), which was extremely resistant to degradation, with a half-life of 69.3 h. It was concluded that both the number of disulphide linkages and tertiary structure were important in determining resistance of proteins to rumen degradation, and that incorporation of SFA 8 and ovalbumin proteins into forages using genetic engineering techniques would be likely to increase the quantity of SAA by passing rumen fermentation.     

Molecular and technological characterization of Staphylococcus xylosus isolated from naturally fermented Italian sausages by RAPD, Rep-PCR and Sau-PCR analysis

Coagulase-negative cocci (CNC) are important microorganisms in fermented sausages because they release lipases and proteases that are able to free short-chain fatty acids and peptides and aminoacids, respectively, that are responsible for the aroma of fermented sausage. The purpose of this study was to characterize Staphylococcus xylosus strains isolated from naturally fermented sausages, produced in three different processing plants in the Friuli Venezia Giulia region in the Northeast of Italy. Two hundred and forty-nine strains of S. xylosus were identified by species-specific PCR and subjected to molecular and technological characterization. RAPD-PCR with primer M13, Rep-PCR using primer (GTG)(5) and Sau-PCR with primer SAG(1) were used for the molecular analysis, while the capability of the strains to grow at different temperatures and in the presence of NaCl and their lipolytic and proteolytic activity were tested in order to define the technological characteristics. The results obtained allowed us to differentiate strains coming from different plants, thereby admitting the presence of strains that are plant-specific.     

Nutrient elements in the herbage of white clover,red clover,lucerne and sainfoin

White clover, red clover, lucerne and sainfoin were grown in the field, and the mineral composition and cation/anion balance of the herbage were determined on samples taken at advancing stages of growth on eight occasions from 29 April to 18 July. The elements which showed the greatest differences in content between species were calcium, sodium, and molybdenum. White clover was consistently higher than the other species in nitrogen, phosphorus, chlorine and molybdenum. Sainfoin was consistently and substantially lower in calcium and sodium than the other species. Evidence based on the total contents of cations and inorganic anions and on determinations of ash alkalinity indicated that white clover and red clover had the highest contents of organic acids, sainfoin having substantially lower, and lucerne intermediate, levels.