Indigenous enzymes in milk: A synopsis of future research requirements

Milk contains a large number of indigenous enzymes, with differing functions, stability to processing, impact on dairy products, and significance for consumer safety (e.g., antimicrobial enzymes). Some enzymes are of interest for their beneficial activity (e.g., lactoperoxidase), some for use as indices of processing (e.g., alkaline phosphatase) and some for effects on the quality of dairy products (e.g., plasmin, lipoprotein lipase), which may be either positive or negative for different products. The study of enzymes in milk is a key specialisation within both the fields of biochemistry and dairy science, and remains an active research subject. Many questions remain to be answered about the nature and significance of milk enzymes, and progress is in some cases hampered by inconsistencies in assay methodologies being used; some of these issues are discussed in this article, the content of which was generated in part by discussions at the First IDF Symposium on Indigenous Enzymes in Milk, Cork, Ireland, 20–22 April 2005.     

Indigenous enzymes in milk: Overview and historical aspects—Part 2

Between 1924 and about 1970, many indigenous enzymes were identified in milk. These were important as indicators of the adequate pasteurisation of milk (alkaline phosphatase, γ-glutamyl transferase) or of mastitis (N-acetylglucosaminidase, acid phosphatase) and some were considered to be important for the stability of milk (superoxidase dismutase, sulphydryl oxidase). Human and equine milk both contain a very high level of lysozyme, which is considered to have a significant protective effect on the neonate. Progress on the isolation and characterisation of these seven enzymes first isolated in the period 1925–1970, as well as ribonuclease, aldolase and glutathione peroxidase, from the milk of the cow and other species and their significance in milk and dairy products is reviewed in this article.     

Indigenous enzymes in milk: Overview and historical aspects—Part 1

The indigenous enzymes in milk have been the subject of research since 1881 when the first report on an indigenous enzyme (lactoperoxidase (LPO)) appeared. These enzymes originate from an animal's blood plasma, leucocytes (somatic cells) and the apical membrane or cytoplasm of the secretory cells. By the early 20th century, seven indigenous enzymes had been identified in milk: LPO, catalase, xanthine oxidase, proteinase, lipase, salolase (arylesterase) and amylase. These were probably the most widely recognised enzymes at that time and, in addition, some of them were relatively easily assayed or were technologically important. The progress of research on these enzymes to the present date will be reviewed in this article.     

Physiological role of indigenous milk enzymes: An overview of an evolving picture

Over 60 indigenous enzymes have been identified so far in the milk of various mammalian species. The vast majority of research in this area has focused on their use as indicators of processing (mainly pasteurization), contribution to dairy product quality and investigating the factors that affect their level in milk. The aim of this article is to provide an overview of data accumulated during the last 5 years, mostly for bovine and human milk, which shows that milk indigenous enzymes play a key role in regulating lactogenesis, e.g., inducing active involution, and that they are essential components of antioxidation and the innate immune system of milk.     

Effects of high pressure on proteolytic enzymes in cheese: relationship with the proteolysis of ewe milk cheese

Ewe milk cheeses were submitted to 200, 300, 400, and 500 MPa (2P to 5P) at 2 stages of ripening (after 1 and 15 d of manufacturing; P1 and P15). The high-pressure-treated cheeses showed a more important hydrolysis of β-casein than control and 2P1 cheeses. Degradation of α_s1-casein was more important in 3P1, 4P1, and P15 cheeses than control and 2P1 cheeses. The 5P1 cheeses exhibited the lowest degradation of α_s-caseins, probably as a consequence of the inactivation of residual chymosin. Treatment at 300 MPa applied on the first day of ripening increased the peptidolytic activity, accelerating the secondary proteolysis of cheeses. The 3P1 cheeses had extensive peptide degradation and the highest content of free amino acids. Treatments at 500 MPa, however, decelerated the proteolysis of cheeses due to a reduction of microbial population and inactivation of enzymes.     

A brief overview of the tobacco industry in the last 20 years

Since the launch of Tobacco Control 20 years ago, there have been several changes in the tobacco industry worldwide. The goal of this commentary is to present some of the keys changes of the past two decades. This time is marked by mergers and acquisitions that led to the existence, today, of four major transnational tobacco companies: Philip Morris International, British American Tobacco, Japan Tobacco and Imperial Tobacco. The possible role of the China National Tobacco Corporation in the world tobacco market is also discussed. In addition, in the past decade there was an increase in tobacco companies' investment in non-cigarette forms of nicotine delivery. The impact of these changes for tobacco control policy is briefly discussed.     

Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.     

Determination of proteolytic activity in different milk systems

A method for the determination of proteolytic activity in whole milk, skim milk or in a solution similar to skim milk ultrafiltrate was optimized and validated. An artificial substrate (azocasein) was used to quantify the enzyme activity through the release of a chromogenic product that was measured spectrophotometrically after clarifying the samples by the addition of trichloracetic acid. The method gave linear results in the range of 0–50 mU/ml of added Bacillus subtilis protease and the least detection and quantification limits were 2.29 and 7.64 mU/ml of protease, respectively. The precision, measured as relative standard deviation, was between 1.14 and 7.99% and mean recovery ranged between 99 and 104%. Reliability of the method was satisfactory for all products evaluated.     

Composition, indigenous proteolytic enzymes and coagulating behaviour of ewe milk as affected by somatic cell count

This study was undertaken to assess the effect of somatic cell count in ewe milk on i) composition and hygienic traits; ii) plasmin, cathepsin and elastase activities; iii) leukocyte differential count; iv) renneting parameters. Individual ewe milk samples were grouped according to somatic cell count (SCC) into five classes: SC300 (<300 000 cells/ml), SC500 (from 301 000 to 500 000 cells/ml), SC1000 (from 501 000 to 1 000 000 cells/ml), SC2000 (from 1 001 000 to 2 000 000 cells/ml) and SC>2000 (>2 001 000 cells/ml). Individual milk samples were analysed for pH, chemical composition, microbial features, indigenous proteolytic enzymes, differential leukocyte population, and renneting parameters. Milk yield, lactose, protein, non casein nitrogen, microbial features were affected by SCC level. Plasmin and elastase activities were the highest in samples with more than 1 000 000 cells/ml; plasmin had intermediate values in samples with 300 000 to 1 000 000 cells/ml and the lowest in samples with less than 300 000 cells/ml of milk. Cathepsin D showed significantly lower values in SC300 and SC1000 classes than in SC500, SC2000 and SC>2000 classes. The highest percentages of lymphocyte were found in samples with less than 1 000 000 cells/ml, while the highest levels of polymorphonuclear leukocyte were found in samples with more than 1 000 000 cells/ml of milk. Longer clotting time was found in SC>2000 samples, while reduced clot firmness was observed in SC500 and SC>2000 samples. Results on milk yield and on compositional parameters evidenced an impairment of udder efficiency in ewe milk samples starting from 300 000 cells/ml. Plasmin activity in milk can be considered as a marker of the synthetic and secreting ability of the mammary gland; furthermore plasmin and elastase were consistent with the health status of the udder. Finally cathepsin D played a role in the worsening of renneting properties of ewe milk.     

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.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

Effects of high pressure treatment on the proteolytic enzymes in meat

This paper describes the effects of high-pressure treatment on proteolytic enzymes in muscle, especially catheptic enzymes which influence meat tenderization, and on acid phosphatase, used as an index of disruption of lysosomal membranes. Acid phosphatase activity in the extract from pressurized muscle increased with increasing pressure applied to the muscle up to 500 MPa. Activity of cathepsin B, D and L increased up to 400 MPa, then tended to decrease at 500 MPa. Cathepsin H and aminopeptidase B decreased with the increasing pressure. Measurements of enzymic activity in the pressurized crude extract, to investigate the pressure effect on the enzymes themselves, showed that all enzymes studied in this paper lost their enzymic activity as applied pressure increased. When the pressurized extracts were subjected to the gel-filtration chromatography, a decrease in the activities of cathepsin H and aminopeptidase B and an increase in the activities of cathepsins B and L and acid phosphatase were observed. It seems that the decrease in activity of the enzymes eluted early from the column (cathepsin H and aminopeptidase B) is due to decrease in the amount of protein eluted by the pressure treatment, whereas the increase in activity of the enzymes eluted late (cathepsin B, L and acid phosphatase) is due to an increase in the amount of protein eluted. From the results, it was concluded that the pressure-induced increase in the amount of protease activity in the muscle was due to the release of the enzymes from lysosomes.     

美国甜菜制糖工业简介

1前言 美国是世界主要食糖出口国之一，制糖生产主要包括甜菜和甘蔗制糖工业，根据美国农业经济研究服务部报告^[1]，甜菜糖约占54％而甘蔗糖占46％。美国的甜菜种植主要分布在五个区域包括2个州，总面积达到160万英亩（960万亩）^[2]，最大的甜菜种植区域是位于北达科他州东部和明尼苏达州东部的红河谷，该区域约占美国甜菜种植面积的50％达73．4万英亩。     

The effect of proteolytic enzymes on the thermostability of egg-white

The effect of proteolytic enzyme preparations such as Proteopol PB (bacterial origin), ficin, and pancreatin on the thermostability of egg-white proteins during pasteurization was investigated. Proteolytic activities of each enzyme were measured at three different doses which were calculated by the ANSON method. The degree of white protein degradation was evaluated by the determination of amino-nitrogen. Effectiveness in reducing the coagulation of egg-white proteins varied, depending on the type and dose of enzyme used. Proteopol PB showed only little effect, ficin demonstrated the best anticoagulation properties at the lowest dose, while for pancreatin the middle dose proved to be the most effective. It was concluded that moderate proteolysis is useful for improving the thermostability of egg-white proteins during pasteurization. Total bacterial counts increased considerably after incubation of egg-white with enzyme preparations, but after pasteurization the number of bacteria decreased to levels which were compatible with bacteriological standards for egg-white products.     

Bioactive peptides in dairy products: synthesis and interaction with proteolytic enzymes

Food proteins are sources of peptides with various biological activities, such as opioids, mineral carriers, antihypertensives, immuno-stimulants, antithrombotics and antigastrics. These bioactive peptides are generated in vivo, in vitro and during food processing. Bioactive peptides are largely found in milk, fermented milks and cheeses. Proteolytic enzymes naturally occurring in milk, and enzymes from lactic acid bacteria or from exogenous sources contribute to the generation of bioactive peptides. Dairy processing conditions such as cheese ripening are also relevant. Once produced, bioactive peptides play a significant role in cheesemaking by selectively inhibiting proteolytic enzymes of lactic acid bacteria and subsequently affecting cheese quality. Sensitivity to inhibition is highly specific: enzymes of the same biochemical class and strains of the same species differ. Bioactive peptides also inhibit dairy spoilage enzymes.     

Oxidative damage to proteolytic enzymes: inactivation of papain and ficin by ferrylmyoglobin

 Ferrylmyoglobin [MbFe(IV)=O], formed by activation of metmyoglobin by hydrogen peroxide, inactivates the cysteine proteinases papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) more efficiently than hydrogen peroxide, but less efficiently than hydroxyl radicals as generated by peroxynitrite or the Fenton reaction. Metmyoglobin and oxymyoglobin could not inactivate papain and ficin. Oxidation of papain and ficin by ferrylmyoglobin occurs in enzyme/haem-protein complexes with binding constants of approximately 10⁵ l · mol^–1; inactivation of proteolysis by papain plateaus at neutral pH to about ¹/₃ whereas the inactivation under acidic conditions was larger. Received: 11 July 1997 / Revised version: 16 September 1997     

Oxidative damage to proteolytic enzymes: inactivation of papain and ficin by ferrylmyoglobin

 Ferrylmyoglobin [MbFe(IV)=O], formed by activation of metmyoglobin by hydrogen peroxide, inactivates the cysteine proteinases papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) more efficiently than hydrogen peroxide, but less efficiently than hydroxyl radicals as generated by peroxynitrite or the Fenton reaction. Metmyoglobin and oxymyoglobin could not inactivate papain and ficin. Oxidation of papain and ficin by ferrylmyoglobin occurs in enzyme/haem-protein complexes with binding constants of approximately 10⁵ l · mol^–1; inactivation of proteolysis by papain plateaus at neutral pH to about ¹/₃ whereas the inactivation under acidic conditions was larger. Received: 11 July 1997 / Revised version: 16 September 1997     

Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk

Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas. The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10(8) CFU/ml of P. fluorescens. With this method it was possible to detect proteolytic bacteria at 10(5) CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification.