Beta-Galactosidase of Streptococcus cremoris H

Beta-galactosidase (Beta-D-galactosidegalactohydrolase, EC 3.2.1.23) of Streptococcus cremoris H was partially purified by ammonium sulfate fractionation. Only 10% of the protein was recovered as enzyme protein and more than 50% of the enzyme in the crude extract was lost. A 2.6-fold purification only was achieved. The enzyme was most active at 65°C and recorded an optimum pH at 7.0. Km and V_max with ortho-nitrophenyl beta-D galactopyranoside as the substrate were recorded as 0.384 mM and 12.6 μmoles/mg protein/min. Manganese ions activated the enzyme. The enzyme was strongly inhibited by Hg⁺⁺, Ca⁺⁺, Ni⁺⁺, and Ag⁺ as well as parachloromercuribenoate.     

Beta-lactamase-producing mutants of Streptococcus cremoris

Penicillin-resistant mutants were isolated for six strains of Streptococcus cremoris used in commercial Cheddar cheese manufacture after treatment with N-methyl-N-nitro-N-nitrosoguanidine. The resistant mutants had an elevated minimal growth inhibitory concentration, 2.5 micrograms (4.13 units)/ml, for penicillin G and other beta-lactam antibiotics as compared with the penicillin-susceptible parent strains, which were each sensitive to .05 micrograms (.08 units)/ml. Penicillin resistance was due to the production of beta-lactamase. Plasmid DNA was not demonstrated in partially purified lysates of four mutants. Mutants had normal cellular morphology but altered phage sensitivity patterns. All except one strain were able to support complete phage adsorption. Resistance was retained after 20 passages in absence of penicillin.     

Thermoinducible Lysis of Temperature-Sensitive Streptococcus cremoris Strains

Temperature-sensitive Streptococcus cremoris SK11 appeared to harbor a temperate phage that was induced at the maximum cooking temperatures used in Cheddar cheese manufacture, i.e., 38 to 40°C. When incubated at 30°C in M17-lactose broth, followed by a shift to 40°C, lysis of the culture occurred within 2 h. Results were similar when S. cremoris SK11 was propagated in M17-lactose broth through a simultated Pearce activity test. In 11% solids reconstituted NDM, this strain also exhibited decreased cell numbers approximately 2 h after the temperature had reached 40°C in the Pearce activity test. To support the hypothesis that lysis of the cells was due to induction of phage, electron microscopy of the temperature-induced lysate revealed the presence of particles resembling phage heads and tails but only a few intact phage-like particles. Examination of other temperature-sensitive or temperature-insensitive strains of S. cremoris also suggested that lysis of temperature-sensitive strains occurred after cells were incubated at 40°C, but no lysis was observed for temperature-insensitive strains.     

RECOVERY OF PROTEINASE FROM PACIFIC WHITING SURIMI WASH WATER

A proteinase from Pacific whiting surimi wash water (SWW) was recovered by ohmic heating, ultrafiltration, and freeze drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. Ohmic heating conditions were optimized for the maximum recovery of the enzyme. Different applied voltages (50, 70, 90 V) showed no differences in efficiency for removing protein and retaining cathepsin Lactivity. Cathepsin L activity reached its maximum after ohmic heating to 55C whereas cathepsin B activity decreased constantly with increased temperature. A constant reduction in protein content was observed with the increase in temperatures from 45C to 60C and holding time up to 5 min. The highest retention of both total and specific activity of cathepsin L was obtained with the treatment at 55C for 3 min. Under these conditions, 193% activity was recovered from SWW although a large amount of the activity was lost by the subsequent steps of ultrafiltration and freeze drying.     

Manufacture of Cheddar Cheese Using Proteinase-Negative Mutants of Streptococcus cremoris

Cheddar cheese was manufactured with a proteinase-negative mutant of Streptococcus cremoris UC 73 and from a commercial lactic culture blend. Soluble nitrogen was analyzed and the cheese graded at intervals to 365 d of age. The cheese made with proteinase-negative cultures graded equal to the control cheese up to 90 d. It was best in overall texture and body and lowest in cheese flavor and flavor intensity after 90 d. It also had significantly higher soluble nitrogen throughout storage. No significant differences in yields were found.Cheddar cheese made at a constant 39°C temperature with a 2% inoculum of proteinase-negative S. cremoris UC 73 increased manufacturing time by 40 min over cheese made by conventional cooking procedures. When inoculum was increased to 4% and the constant temperature to 42°C, manufacturing time was 3.6 h, which was 1 h less than with a 2% inoculum and conventional cooking. By providing a yeast extract carry-over of .0175% from the proteinase-negative bulk culture, it would be possible to produce Cheddar cheese within a normal time frame with only .7% inoculum.     

CHARACTERIZATION OF PROTEINASE RECOVERED FROM PACIFIC WHITING SURIMI WASH WATER

Pacific whiting surimi wash water (SWW) proteinase was recovered by ohmic heating, ultrafiltration, and freeze-drying. By these processes, 5.9-fold purification was achieved. The most efficient step was ohmic heating, which concentrated the proteinase by 4.8 fold. Specific activity of the recovered SWW proteinase on casein and Z-Phe-Arg-NMec was 28.2 and 0.17 U/mg protein, respectively. The SWW proteinase showed good hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. Acidification increased specific activity on all substrates tested but reduced thermal stability. β-Mercaptoethanol, dithiothreitol and urea enhanced activity against Z-Phe-Arg-NMec. Proteinase activity on Z-Phe-Arg-NMec showed an optimum pH of 4.0. The recovered proteinase showed 18.5% residual activity after 7 week storage at 4C.     

FRACTIONATION AND CHARACTERIZATION OF CYSTEINE PROTEINASE INHIBITOR FROM CHICKEN PLASMA

The fractionation of cysteine proteinase inhibitor (CPI) from chicken blood plasma was carried out using polyethylene glycol‐4000 or ammonium sulfate (AS) precipitation. The addition of PEG at the level of 400 g/L, on the basis of the original volume of plasma protein, was more effective to fractionate CPI than using AS. CPI in the PEG fraction had a molecular weight of about 46 kDa with intramolecular disulfide bond. CPI containing fraction was colorless and had no absorbance in the range of 700–360 nm. The fraction was stable in the temperature range of 40–90C for 10 min and still retained high inhibitory activity toward papain after incubation at 90C for 60 min. NaCl, at 0–3.0% concentration, did not affect the inhibitory activity of the CPI containing fraction. The fraction was stable at pH 8.0, and the minimal inhibitory activity against papain was found at pH 5–6. Therefore, PEG fractionation effectively isolated the CPI from chicken plasma.     

Effect of Proteolytic Activity of Streptococcus cremoris on Cottage Cheese Yields

Using only a proteinase-negative variant of Streptococcus cremoris UC310 to manufacture cottage cheese increased theoretical yields by 2.26% compared wth the proteinase-positive parent. Yield differences between positive and negative variants were strain dependent and not detected with variants of UC73 and UC97. Growth of proteinase-negative variants in bulk culture required pH control and addition of sufficient nitrogenous stimulant to provide carry-over activity into the cheese milk. Cultures developed normally when the bulk medium contained 5% of a blend of yeast extract and casein hydrolyzate. Proteinase-negative culture successfully completed acidification of cottage cheese milk after direct acidification to pH 5.2 with phosphoric acid. Lactic culture strain selection is suggested for maximizing product yields with proteinase-negative cultures.     

Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis.

Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The additon of Mn2+ stimulated growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c.1000 and 600 mug/g in the case of Leuc. cremoris FR8-1 and CAF1, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.     

Effects of Antibiotics on Proteinase-Positive and Proteinase-Negative Variants of Streptococcus cremoris

The growth and acid-producing ability of proteinase-positive and proteinase-negative starter cultures were studied in the presence of several antibiotics commonly used in treating bovine mastitis. Proteinase-positive and proteinase-negative variants of two different strains of Streptococcus cremoris were inoculated separately into heat-sterilized, reconstituted nonfat milk containing a range of concentrations of either dihydrostreptomycin, erythromycin, or penicillin G. Generation times and pH changes were determined for each sample following incubation at 38°C for 5 h.At 2% inoculation, proteinase-positive cultures produced more pH change than their proteinase-negative counterparts at all concentrations of all three antibiotics. Proteinase-negative cultures at 8% inoculation produced more acid than proteinase-positives did when inoculated at 2%. Generation times for proteinase positives and proteinase negatives were the same when both cultures were inoculated at 2%, but 8% inoculation of proteinase negatives produced significantly shorter generation times. Increases in antibiotic concentrations affected proteinase-positives more than proteinase-negatives in both pH change and generation time. Penicillin affected both proteinase positives and negatives more than the other two antibiotics did but affected positives more than negatives.     

PROPERTIES OF CYSTEINE PROTEINASE INHIBITORS FROM BLACK GRAM AND RICE BEAN

Cysteine proteinase inhibitors (CPI) were purified to 59 and 54 fold from black gram (Vignaraungo (L.) Hepper) and rice bean (Vignaumbellata Thunb.), respectively, by using heal treatment, followed by chromatography on a carboxymethyl (CM)‐papain‐Sepharose affinity column. The purified inhibitors were highly inhibitory to papain and Pacific whiting cathepsin L in a concentration dependent manner. They were detected as a dark band on tricine‐SDS‐PAGE gel stained for inhibitory activity. The apparent molecular weights of purified CPI from black gram and rice bean seeds were estimated to be 12, 000 daltons. The purified inhibitors were thermostable up to 90C and active in the neutral and alkaline pH ranges.     

USE OF CYSTEINE PROTEINASE INHIBITORS FROM INJURED TOMATO LEAVES IN WHITING SURIMI

Cooking surimi paste from Pacific whiting results in a gel with poor texture due mainly to myosin degradation caused by a cysteine proteinase. Cysteine and serine proteinase inhibitors were isolated from injured and methyl jasmonate treated tomato leaves. Tomato cysteine proteinase inhibitor was stable at 60C but inactivated at 90C, making it suitable for use in surimi. Tomato proteinase inhibitors (TPI), having 7.9 papain inhibitor units, inhibited autolysis about 95% in 10 g of Pacific whiting surimi. Gel strength of Pacific whiting surimi was improved by adding only 0.0 27% of TPI to the surimi formulation. Addition of TPI did not affect the color of whiting surimi gel, while egg white needed to prevent gel weakening caused the gels to have more yellow hue (P<0.05). SDS-PAGE showed that myofibrillar protein degradation was prevented during cooking when 0.027% of TPI was included in the surimi. TPI extracted from tomato plants has potential for use as food grade additive in Pacific whiting surimi.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

Changes in populations of Listeria monocytogenes in a medium with internal pH control containing Streptococcus cremoris

Behavior of Listeria monocytogenes in a commercial starter culture medium with internal pH control was examined. The IPCM-1 medium was inoculated with L. monocytogenes (strain V7, Scott A, or California) at ca. 10(3) cfu/ml and Streptococcus cremoris (.25%, 1.6 x 10(5) or 1.0%, 8.6 x 10(5) cfu/ml) and was incubated at 21 or 30 degrees C for 30 h. The pH of the uninoculated medium and control (L. monocytogenes only) was between 6.8 and 7.0 before and after incubation. The area on a figure between control and treatment curves for numbers of L. monocytogenes was calculated, designated as area of inhibition, and used to quantitate inhibition of L. monocytogenes caused by S. cremoris. No significant difference was found in area of inhibition or pH values at 6, 24, and 30 h of incubation among the three strains of L. monocytogenes for a given set of conditions (percentage of S. cremoris added and temperature). Inhibition of the pathogen increased with an increase in amount of S. cremoris inoculum used and with higher rather than lower incubation temperature. Populations of L. monocytogenes in IPCM-1 medium without S. cremoris after 30 h of incubation were 10(6) to 10(7)/ml at 21 degrees C and 10(8)/ml at 30 degrees C. At 21 degrees C inhibition of Listeria began after 18 and 24 h and at 30 degrees C after 12 and 15 h of incubation in samples inoculated with 1.0 and .25% S. cremoris, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE INHIBITOR FROM TEPARY BEAN (PHASEOLUS ACUTIFOLIUS) SEEDS

In a qualitative screening of 36 accessions of tepary beans seeds, all the accessions inhibit the activity of bovine trypsin and trypsin-like proteinases from the insect P. truncatus, and the majority of them inhibit α-amylase activity of several important insect pests. A protein proteinase inhibitor was purified from accession L-242-45, using fractional precipitation, gel filtration, ion exchange chromatography and reverse-phase HPLC. The protein showed an apparent molecular weight of 7,100 by PAGE. However, contrary to other inhibitors previously reported, the inhibitory activity was only present in the trimeric form. The protein was characterized as a serine-proteinase inhibitor that recognized trypsin, chymotrypsin and trypsin-like proteinases, but it also recognized aspartic acid proteinases from different insects. It contained no carbohydrate residues and showed a high stability at 96C at low pH.     

Plasmid heterogeneity in Streptococcus cremoris M12R: effects on proteolytic activity and host-dependent phage replication

Examination of single colony isolates from a culture of Streptococcus cremoris M12R revealed a high degree of variability in plasmid deoxyribonucleic acid composition. Fifty percent of the M12R population displayed proteolytic activity and harbored a 13-Mdalton plasmid (pLR2013). This plasmid was not present in proteinase-deficient variants isolated from the culture, which provided correlative evidence for linkage of proteinase activity to pLR2013. Four percent of the M12R population demonstrated resistance to phage m12r X M12. This resistance was identified by restriction and modification activities against m12r X M12 phage, which was dependent on the presence of a 20-Md plasmid, pLR1020. Loss of restriction and modification activities was observed upon curing of pLR1020. In conjugal mating studies with Streptococcus lactis ME2, transfer frequency of lactose-fermenting ability to a restriction and modification-deficient variant of M12R was 10(2)-fold higher than to a variant exhibiting restriction and modification activities. The data provided evidence for restriction and modification activities in select S. cremoris M12R variants that are linked to pLR1020 and restrict both the plaquing ability of phage and efficiency of plasmid transfer by conjugation.