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In 2020, nearly 3 million scientific and engineering papers were published worldwide (White, K. Publications Output: U.S. Trends And International Comparisons). The vastness of the literature that already exists, the increasing rate of appearance of new publications, and the timely translation of artificial intelligence methods into scientific and engineering communities have ushered in the development of automated methods for mining and extracting information from technical documents. However, domain-specific approaches for extracting knowledge graph representations from semantic information remain limited. In this paper, we develop a natural language processing (NLP) approach to extract knowledge graphs resulting in a semantically structured network (SSN) that can be queried. After a detailed exposition of the modeling method, the approach is demonstrated specifically for the synthetic chemistry of organic molecules from the text of approximately 100,000 full-length patents. In this paper, we focus specifically on characterizing the knowledge graph to develop insights into the linguistic patterns and trends within the data and to establish objective graph characteristics that may enable comparisons among other text-based knowledge graphs across domains. Graph characterization is performed for network motif structures, assortativity, and eigenvector centrality. The structural information provided by the measures reveals language tendencies commonly employed by authors in the text discourse for chemical reactions. These include observations of the prevalence of descriptions of specific compound names, that common solvents and drying agents cut across large numbers of chemical synthesis approaches, and that power-law trends clearly emerge in the limit of larger corpora. The findings provide important quantitative characterizations of knowledge graphs for use in validation in large data settings.

Recent studies highlight a persistent increase in subsequent injury risk following a sport-related concussion (SRC) despite clinical recovery. However, markers of persistent alterations in sensorimotor integration have yet to be identified. One possibility is that compensatory adaptation following SRC may only be unmasked during transient periods of high task complexity in specific sensorimotor circuits. The current study used short-latency afferent inhibition (SAI) to investigate the long-term sequelae of sport-related concussion (SRC) in different short-latency sensorimotor circuits converging in the motor cortex. Specific sensorimotor circuits sensitive to posterior-anterior current with a positive phase lasting 120µs (PA 120 ) and anterior-posterior current with a positive phase lasting 30µs (AP 30 ) were assessed using controllable pulse parameter transcranial magnetic stimulation (cTMS) while young adults with and without a history of SRC were at rest or responded to valid and invalid sensorimotor cues. SAI was quantified as the ratio of the motor-evoked potential (MEP) elicited by peripherally conditioned cTMS stimuli to the unconditioned MEP for each cTMS configuration. Individuals with a SRC history demonstrated persistent adaptation in AP 30 SAI, but only in response to invalid cues. Persistent adaptation in AP 30 SAI was not apparent at rest or during simple sensorimotor transformations in response to valid cues. PA 120 SAI demonstrated similar responses at rest and in response to both valid and invalid cues, regardless of SRC history. AP 30 -sensitive sensorimotor circuits may mark the long-term SRC sequelae and the increased susceptibility to momentary breakdowns in sensorimotor integration during periods of high cognitive-motor demands., Competing Interests: Declarations. Ethical approval: The procedures used in this study adhere to the tenets of the 1964 Declaration of Helsinki. The Clinical Research Ethics Committee at the University of Waterloo approved the study protocol (ORE #43252). Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: Not applicable. Conflict of interest: The authors declare no competing financial or non-financial interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Skilled motor ability depends on efficiently integrating sensory afference into the appropriate motor commands. Afferent inhibition provides a valuable tool to probe the procedural and declarative influence over sensorimotor integration during skilled motor actions. This manuscript describes the methodology and contributions of short-latency afferent inhibition (SAI) for understanding sensorimotor integration. SAI quantifies the effect of a convergent afferent volley on the corticospinal motor output evoked by transcranial magnetic stimulation (TMS). The afferent volley is triggered by the electrical stimulation of a peripheral nerve. The TMS stimulus is delivered to a location over the primary motor cortex that elicits a reliable motor-evoked response in a muscle served by that afferent nerve. The extent of inhibition in the motor-evoked response reflects the magnitude of the afferent volley converging on the motor cortex and involves central GABAergic and cholinergic contributions. The cholinergic involvement in SAI makes SAI a possible marker of declarative-procedural interactions in sensorimotor performance and learning. More recently, studies have begun manipulating the TMS current direction in SAI to tease apart the functional significance of distinct sensorimotor circuits in the primary motor cortex for skilled motor actions. The ability to control additional pulse parameters (e.g., the pulse width) with state-of-the-art controllable pulse parameter TMS (cTMS) has enhanced the selectivity of the sensorimotor circuits probed by the TMS stimulus and provided an opportunity to create more refined models of sensorimotor control and learning. Therefore, the current manuscript focuses on SAI assessment using cTMS. However, the principles outlined here also apply to SAI assessed using conventional fixed pulse width TMS stimulators and other forms of afferent inhibition, such as long-latency afferent inhibition (LAI).

Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.

With the growing opioid epidemic, plastic surgeons are being encouraged to transition away from reliance on postoperative opioids. However, many plastic surgeons hesitate to use nonopioid analgesics such as nonsteroidal antiinflammatory drugs and local anesthetic blocks because of concerns about their safety, particularly bleeding. The goal of this systematic review is to assess the validity of risks associated with nonopioid analgesic alternatives. A comprehensive literature search of the PubMed and MEDLINE databases was conducted regarding the safety of opioid alternatives in plastic surgery. Inclusion and exclusion criteria yielded 34 relevant articles. A systematic review was performed because of the variation between study indications, interventions, and complications. Thirty-four articles were reviewed that analyzed the safety of ibuprofen, ketorolac, celecoxib, intravenous acetaminophen, ketamine, gabapentin, liposomal bupivacaine, and local and continuous nerve blocks after plastic surgery procedures. There were no articles that showed statistically significant bleeding associated with ibuprofen, celecoxib, or ketorolac. Similarly, acetaminophen administered intravenously, ketamine, gabapentin, and liposomal bupivacaine did not have any significant increased risk of adverse events. Nerve and infusion blocks have a low risk of pneumothorax. Limitations of this study include small sample sizes, different dosing and control groups, and more than one medication being studied. Larger studies of nonopioid analgesics would therefore be valuable and may strengthen the conclusions of this review. As a preliminary investigation, this review showed that several opioid alternatives have a potential role in postoperative analgesia. Plastic surgeons have the responsibility to lead the reduction of postoperative opioid use by further developing multimodal analgesia.

Medical tourism, or traveling abroad to obtain medical services, has evolved into a global health care phenomenon, with over 15 million U.S. patients each year seeking medical care internationally, representing a $50 billion dollar industry in 2017. Speculation and media fascination about the growing industry, diverse destinations, and rationale behind the medical tourists is rampant; however, the legal implications of tourism medicine, particularly when it goes wrong, are often unclear. On the international stage, accreditation agencies are limited in scope and practice, legal jurisdiction is difficult to establish, and the enforcement of rulings is nearly impossible. Patients seeking tourism medicine have little legal recourse and shoulder all the legal burden.

Background: Psychoactive drug use is on the rise in the United States, with plastic surgery patients a potentially susceptible group. This study aimed to determine the incidence of cosmetic and reconstructive patients in our practice taking psychoactive drugs and to compare those values with the national average. Furthermore, we discuss the patient safety concerns when patients withhold their medical history information over the course of their treatment., Methods: Urban private plastic practice patients who underwent surgery in a closed practice from 2009 to 2016 were divided into cosmetic and reconstructive cohorts. Review for drug use was medical scripts, history, and Surescripts drug reporting. Extracted information includes age, race, procedure, psychoactive medications, and whether or not they stated a mental health diagnosis on their medical history forms. Only patients with complete records were included., Results: A total of 830 patients were included in statistical analysis. Due to minimal cohort number, 70 men were excluded, as there were no comparative national data. Our analysis found that 33.6% cosmetic patients and 46.3% reconstructive patients used at least one psychoactive drug., Conclusion: There is a statistically significant difference between psychoactive drug use at our practice compared with the general population and a significantly larger percentage of reconstructive patients taking drugs compared with the cosmetic cohort.

Objective: To evaluate a cohort of gynecologic oncology patients to discover risk factors for early- and late-occurring incisional hernia after midline incision for ovarian cancer., Methods: We collected retrospective data from patients undergoing primary laparotomy for ovarian cancer at the University of Wisconsin Hospitals and Clinics from 2001 to 2007. Patient characteristics and potential risk factors for hernia formation were noted. Physical examination, abdominal computerized assisted tomography scans, or both were used to detect hernias 1 year after surgery (early hernia) and 2 years after surgery (late hernia)., Results: There were 265 patients available for the 1-year analysis and 189 patients for the 2-year analysis. Early and late hernia formation occurred in 9.8% (95% confidence interval [CI] 6.2-12%) and an additional 7.9% (95% CI 4.1-12%) of patients, respectively. Using multiple logistic regression, poor nutritional status (albumin less than 3 g/dL) and suboptimal cytoreductive surgery (1 cm or greater residual tumor) were significantly associated with the formation of early incisional hernia after midline incision (P<.001 for both). Late hernia formation was associated only with age 65 years or older (P=.01)., Conclusion: The formation of early incisional hernias after midline incision is associated with poor nutritional status and suboptimal cytoreductive surgery, whereas late hernia formation is associated with advanced age.

Hydrolysates from yellow stripe trevally meat with 15% degree of hydrolysis (DH) obtained using Alcalase (HA) and Flavourzyme (HF) following pH-stat method were characterized. Both hydrolysates, HA and HF, contained glutamic acid and glutamine as the major amino acids, while glycine was the dominant amino acid in fish flesh. Hydrolysates were also rich in aspartic acid, asparagine, alanine, lysine, and leucine. Essential amino acid/nonessential amino acid ratios were 0.55, 0.63, and 0.62 for flesh, HA, and HF, respectively. After separation on a Sephadex G-50 column, antioxidative activity of hydrolysate fractions was determined as Trolox equivalent antioxidant capacity. The fraction of HF with molecular weight (MW) of 1.77 kDa exhibited the strongest antioxidative activity, compared with other fractions. Among all fractions of HA, the one having a MW of 2.44 kDa showed the highest antioxidative activity. Thereafter, the selected Sephadex G-50 fractions from both HA and HF were further chromatographed using a Sephadex C-25 column, followed by HPLC. MW of antioxidative peptides from HA and HF determined by Maldi TOF/TOF was 656 and 617 Da, respectively. However, peptides isolated from both HA and HF possessed a lower antioxidative activity than Trolox at the same concentration (P < 0.05). HA and HF prevented DNA oxidative damage in Fenton reaction system tested by in vitro plasmid DNA relaxation assay. These results indicate that hydrolysates from yellow stripe trevally can be used as an important source of amino acids and serve as alternative natural antioxidants.

The effect of processing temperature and pH as well as enzyme-assisted extraction on the content and profile of isoflavones in a soymilk system was investigated. Isoflavone content in thermally treated soymilk at pH 7 and pH 9 was determined following a standard solvent extraction or an enzyme-assisted extraction protocol. Upon thermal processing, at both pH 7 and pH 9, significant interconversions were noted, indicated by the observed decrease in malonylglucosides with the concurrent increase in beta-glucosides. Enzyme-assisted extraction resulted in enhanced isoflavone extraction efficiency and revealed significant loss in total isoflavone content upon processing. This observation suggested that protein-isoflavone interactions, which are dependent on the protein structure and isoflavone form, affect isoflavone extractability, leading to underestimation of any loss that might have occurred in previously reported thermal studies. Accurate isoflavone measurements are essential to determine the processing conditions that result in the least loss of the biologically relevant isoflavone content.

The conversion and degradation of malonylglucosides were kinetically characterized under elevated pH/heat conditions. Malonylgenistin and malonyldaidzin were heated at 60, 80, and 100 degrees C and pH values of 8.5, 9, and 9.5. A simple kinetic model was developed, which adequately predicted the conversion and degradation reactions. The conversion and degradation rates increased as temperature and pH increased. The rates of conversion of both malonylglucosides into their respective beta-glucosides were comparable under all pH/heat treatments. However, at 100 degrees C, the rates of degradation of malonyldaidzin were approximately double those of malonylgenistin, under all pH treatments. When malonlydaidzin was heated at 100 degrees C and pH 9.5, degradation of the produced daidzin occurred. Therefore, an alternative kinetic model was developed to better predict the conversion and degradation of malonyldaidzin occurring at 100 degrees C and pH 9.5. The models developed provide soy food manufacturers with guidelines for better control of the profile and level of isoflavones..

Isoflavones occur primarily as glycosides (namely, malonyl-, acetyl-, and non-conjugated beta-glycosides) and a small percentage as the bioactive aglycon. The different chemical structures of isoflavones can dictate their stability during processing. Therefore, our objective was to determine the effects of pH and thermal treatments on conjugated isoflavones with regard to interconversions and loss. Conjugated daidzin and genistin were heated at 25, 80, and 100 degrees C under neutral, acidic, and basic conditions. Changes in isoflavone derivatives were monitored using high-performance liquid chromatography. Along with interconversions, considerable loss in total known isoflavone derivatives was noted for each isoflavone, especially under elevated pH and temperature. The malonylglycosides showed more stability than acetylglycosides, especially under acidic conditions. Overall, loss in isoflavone derivatives was significantly higher for daidzin than for genistin glycoside forms. Our results highlighted the significance of chemical structure with regard to stability, which is a key factor in determining soy processing conditions.

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.

A fluorescent labeling method was developed to study plasminogen (PG) concentration and location in simulated bovine milk. Activity and stability of PG labeled with Alexa Fluor 594 (PG-594) were comparable to those of native PG. The fluorescent signal of PG-594 exhibited pH, temperature, and storage stability, and remained stable throughout typical sample treatments (stirring, heating, and ultracentrifugation). These characteristics indicate broad applicability of the fluorescent labeling technique for milk protease characterization. In an example application, PG-594 was added to simulated milk samples to study effects of heat and beta-lactoglobulin (beta-LG) on the distribution of PG. Before heating, about one-third of the PG-594 remained soluble in the whey fraction (supernatant) whereas the rest became associated with the casein micelle. Addition of beta-LG to the system slightly shifted PG-594 distribution toward the whey fraction. Heat-induced PG-594 binding to micelles in whey-protein-free systems was evidenced by a decrease of PG-594 from 31 to 15% in the whey fraction accompanied by an increase of PG-594 from 69 to 85% in casein micelle fractions. When beta-LG was present during heating, more than 95% of PG-594 became associated with the micelle. A comparison with the distribution pattern of PG-derived activities revealed that heat-induced PG binding to micelles accompanies heat-induced PG inactivation in the micelle fraction. Incubation of the casein micelles with the reducing agent beta-mercaptoethanol revealed that disulfide bonds formed between PG and casein or between PG and casein-bound beta-LG are the mechanisms for heat-induced PG binding to casein micelles. Western blotting and zymography results correlated well with fluorescent labeling studies and activity studies, respectively. Theoretically important findings are: 1) when heated, serum PG is capable of covalently binding to micellar casein or complexing with beta-LG in whey and then coadhering to micelles, and 2) PG that associated with micellar casein through lysine binding sites before heating is capable of developing heat-induced disulfide bonds with casein. The overall results are PG covalently binding to micelles and inactivation thereafter. Our results suggest that, instead of thermal denaturation through irreversible unfolding, covalent bond formation between PG and other milk proteins is the mechanism of PG inhibition during thermal processing.

Heat-stable proteases produced by the psychrotroph Pseudomonas fluorescens M3/6 have been shown to affect the plasmin system in milk, which in turn will affect the quality of processed milk. The M3/6 proteases cause dissociation of plasmin from casein in minimally processed milk. The objective of this work was to study the effect of M3/6 protease on the plasmin system, as well as its role in plasminogen activation, under commonly applied cheese-making conditions. Isolated M3/6 protease was added to raw milk, which then was pasteurized, and subjected to pH adjustments and CaCl2 addition. Casein and whey fractions were separated by chymosin treatment then analyzed for plasmin activity. Individual and interaction effects of M3/6 protease addition, pH treatment, and CaCl2 addition on plasmin activity were studied. Enzyme activity assays were carried out to study individually the effect of M3/6 protease on plasmin system components. Kinetic parameters were calculated to characterize the effect of M3/6 protease on plasminogen activation. Plasmin activity increased in the curd fractions of the protease-treated milk that was subjected to conditions most resembling cheese-making conditions, indicating that M3/6 protease triggered plasminogen activation rather than dissociation of plasmin from casein micelles. Results from the studies on plasminogen activation confirmed that the observed activation of plasminogen in protease-treated samples subjected to cheese making conditions was attributed to the stimulatory effect M3/6 protease had on plasminogen activators (PA). The M3/6 protease stimulated human and bovine PA by increasing their activity 4.5- and 2.5-fold, respectively. Similarly, the catalytic efficiencies of human urokinase-type PA and bovine PA were increased in the presence of M3/6 protease by 12- and 4-fold, respectively. Our research presented a basic step toward fully understanding the effect of bacterial proteases under different processing conditions, where the gathered information can aid in better control of processing conditions based on the desired outcome.

The etiologies of a variety of skin conditions associated with diabetes have not been fully explained. One possible etiological factor is diabetic microangiopathy, which is known to affect the eyes and kidneys in patients with diabetes. There are many mechanisms by which diabetes may cause microangiopathy. These include excess sorbitol formation, increased glycation end products, oxidative damage, and protein kinase C overactivity. All of these processes occur in the skin, and the existence of a cutaneous diabetic microangiopathy has been well demonstrated. These microangiopathic changes are associated with abnormalities of skin perfusion. Because the skin plays a thermoregulatory role, there is significant capillary redundancy in normal skin. In diabetic patients, loss of capillaries is associated with a decrease in perfusion reserve. This lost reserve is demonstrable under stressed conditions, such as thermal stimulation. The associated failure of microvascular perfusion to meet the requirements of skin metabolism may result in diverse skin lesions in patients with diabetes. Many skin conditions peculiar to diabetes are fairly rare. Necrobiosis lipoidica diabeticorum (NLD) and diabetic bullae occur very infrequently as compared with diabetic retinopathy and nephropathy. Conversely, there is a correlation between diabetic microvascular disease and NLD. This correlation also exists with more common skin conditions, such as diabetic dermopathy. This relationship suggests that diabetic microangiopathy may contribute to these conditions even if it is not primarily causal. Clinically, the major significance of diabetic cutaneous microangiopathy is seen in skin ulceration which is very common and has a major impact on diabetic patients. Many factors contribute to the development of diabetic foot ulcers. Neuropathy, decreased large vessel perfusion, increased susceptibility to infection, and altered biomechanics all play a role, but there is no doubt that inadequate small blood vessel perfusion is a major cause of the inability to heal small wounds that eventually results in ulcer formation. The accessibility of skin capillaries makes cutaneous diabetic microangiopathy an attractive model for research on the evolution of microvascular disease in diabetic patients.

The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), and BSA on plasminogen activators. Plasminogen activator activity was significantly stimulated by non-heat treated and denatured alpha-LA as well as by denatured beta-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.

Human plasminogen undergoes a large tertiary structural change in the presence of lysine derivatives (e.g. epsilon-amino caproic acid, EACA). This change facilitates human plasminogen activation by human plasminogen activators, resulting in elevated blood plasmin levels. It is hypothesized that this structure-function relationship is similar for bovine plasminogen. The objectives of this study were to investigate the effect of the ligand EACA on the secondary structure of plasminogen (bovine, human, and rabbit) and the tertiary structure of bovine plasminogen using Fourier-transform infrared spectroscopy (FTIR). Spectra of plasminogen, EACA, and a mixture of plasminogen and EACA in water and deuterium were collected using FTIR. Fourier-self deconvoluted spectra in the amide I region (1700-1600 cm(-1)) were used to detect changes in secondary structure of plasminogen after EACA addition. Change in bovine plasminogen tertiary structure was determined by comparing ratios of amide II (1600-1500 cm(-1)) to amide I bond intensities over time for samples in deuterium. No differences in secondary structure were observed for any plasminogen in the presence of EACA; however, addition of EACA significantly changed tertiary structure of bovine plasminogen. This tertiary structural change indicates a transition from a folded to an unfolded state, which could be more easily converted to plasmin. These results are consistent with reported human plasminogen studies using neutron scattering (tertiary structure) and circular dichroism (secondary structure) methods.

In the present study, we investigated the effect of Pseudomonas spp. growth on the plasmin enzymatic system in casein and whey fractions of fresh milk. Two bacterial strains, Pseudomonas spp. SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into fresh milk and incubated at 7 degrees C for 3 d. Bacterial counts were approximately 10(8) cfu/ml by d 3. Samples collected every 24 h were treated to separate the casein from the whey fraction. Casein and whey fractions were subjected to electrophoresis to visualize protein breakdown and plasmin activity and to colorimetric assays to quantify plasmin-related activities. With psychrotrophic bacterial growth, plasmin levels in casein fractions decreased significantly and in whey fractions increased then decreased significantly. Fresh milk results were similar for the two strains and were similar to earlier results with reconstituted nonfat dry milk. A transmission electron microscopy study by immunocytochemistry showed the presence of plasmin in casein micelles and its disappearance upon microbial growth in the milk. We hypothesized that extracellular microbial proteases produced by psychrotrophic microorganisms are responsible for this effect. To confirm this, an extracellular bacterial protease was isolated from Pseudomonas fluorescens M3/6 by ammonium sulfate fractionation and ion-exchange chromatography and incubated with fresh milk. Milk samples analyzed during incubation with the protease had significantly increased plasmin and plasminogen activities in the whey fraction within 5 h of incubation, while differences in activities in the casein fraction occurred at time 7.5 h for plasmin activity and 10 h of incubation for plasminogen activity. These quantitative data were supported by plasmin activity as visualized by casein-SDS-PAGE. These results suggest that growth of the Pseudomonas strains in fresh milk, and particularly their production of extracellular proteases, may be a causative factor in the release of plasmin from the casein micelle. Such plasmin release could affect the quality of cheeses and other food products that utilize dairy ingredients.

Growth of psychrotrophic bacteria in nonfat dry milk at refrigeration temperatures was shown previously in our laboratory to cause a shift in plasmin (a native milk protease) from the casein to the whey fraction. The whey fraction from cheesemaking is commonly used to make whey protein concentrates and isolates, which then are used as functional ingredients in various food systems. Plasmin activity in whey protein products may cause breakdown of food proteins to have desirable or undesirable effects on food quality. This raised questions about the level of plasmin in commercial whey protein products and factors that affect this plasmin level. Therefore, the objectives of this study were to determine: 1) plasmin concentrations in sweet and acid whey protein products as influenced by Pseudomonas growth during storage of fresh milk, and 2) plasmin concentrations in commercial whey protein products. Whey type (sweet or acid) had a significantly (P < 0.05) greater effect on whey-associated plasmin activity than did Pseudomonas fluorescens M 3/6 growth. Acid whey protein products had significantly (P < 0.05) higher plasmin concentrations than sweet whey. Plasmin activities associated with acid and sweet whey protein products were both significantly (P < 0.0001) affected by the growth of Pseudomonas fluorescens M 3/6. The interaction effect between bacterial growth and whey type on plasmin activity was not significant (P = 0.2457). Plasmin activity in the reconstituted commercial whey protein concentrates (i.e., sweet and acid) varied considerably (16.3 to 330 micrograms/g of protein), but was significantly lower (2.1 to 4.4 micrograms/g of protein, P < 0.05) in whey isolates. These quantitative data were supported by plasmin activity visualized by casein SDS-PAGE.