Activity Of Lactoperoxidase System Research Articles

Activation of lactoperoxidase system and its potential for microbial inhibition and preservation of milk in the Great African Rift Valley climate

Activation of lactoperoxidase system and its potential for microbial inhibition and preservation of milk in the Great African Rift Valley climate

Activation of lactoperoxidase system in buffalo milk using dual enzyme (lactase & glucose oxidase) and its effect on milk constituents

In the present investigation, lactoperoxidase (LP) system was activated in buffalo milk using 400 U of lactase with 350 U of glucose oxidase as the source of in situ hydrogen peroxide with 50 mg thiocyanate per litre of milk. On the basis of titratable acidity, clot on boiling, and alcohol test, milk remained acceptable up to 9–10 h. The hypothiocyanate ions level (57.629 ± 0.38mM) after 9th hour of LP activation maintained close to the level suggested in central composite rotatable design. The milk preserved using the standardised formulation to activate the LP system did not show any variation in physico‐chemical parameters of milk from the usual quality except for lactose content, which was measured using HPLC. Lactose content was reduced from initial 4.7% to 1.4%, 0.99% and 0.53% after 1, 2 and 4 h of activation at 37°C, respectively. Whey proteins remained unaffected as no changes were observed in their electrophoretic behaviour. Serine equivalents in both control and LP system–activated milk was 1.20 ± 0.001 mg/mL, indicating no proteolysis. Thiobarbituric acid values of LP‐activated milk at 0th hour and 9th hour were 0.81 ± 0.002 and 0.082 ± 0.003, respectively. No significant changes in free fatty acids were observed on account of LP‐system activation using the studied dual‐enzyme approach. LP‐activated milk remained heat stable up to 10 h at 37°C.

Quality criteria of Tulum cheese produced from cow’s milk preserved by activation of lactoperoxidase system

The quality criteria of Tulum cheese produced from cow's milk and preserved by the activation of the lactoperoxidase system (LP-s) were investigated during the 90-d ripening period. Samples of milk containing 20:20 ppm (C), 60:60 ppm (D), and 0:0 ppm (B) SCN:H2O2 were stored at 25°C for 8 hr. As a control (A), one portion of the milk without SCN:H2O2 was stored at 4°C for 8 hr. Total dry matter, fat, salt, total nitrogen (TA), water-soluble nitrogen (WSN), titratable acidity (% L.A), pH, ripening index, volatile fatty acids, tyrosine, acid value, free fatty acids, total bacteria, and coliform bacteria contents were determined after 1, 30, 60, and 90 d of storage. The effect of the treatments on titratable acidity, tyrosine, volatile fatty acid, acid value, free fatty acids, total and coliform bacteria was statistically significant. LP-s treatment did not affect fat, dry matter, total nitrogen water-soluble nitrogen contents of cheese. Practical applications The raw milk preserved by adding 20:20 ppm SCN:H2O2 was suitable for producing Tulum cheeses based on the technological processes and quality characteristics of the product, and was concluded that milk with 60:60 ppm SCN:H2O2 can also be used for the production of this kind of cheese.

Preservation of Raw Camel Milk by Lactoperoxidase System Using Hydrogen Peroxide Producing Lactic Acid Bacteria

This study was conducted to investigate the effect of lactic acid bacteria (LAB) activated lactoperoxidase system (LPs) on keeping quality of raw camel milk at room temperature. Camel milk samples were collected from Errer valley, Babile district of eastern Ethiopia. The level of hydrogen peroxide (H2O2) for activation of LPs was optimized using different levels of exogenous H2O2. Strains of LAB (Lactococcus lactis 22333, Weissella confusa 22308, W. confusa 22282, W. confusa 22296, S. Infatarius 22279 and S. lutetiensis 22319) with H2O2 producing properties were evaluated, and W. confusa 22282 was selected as the best strain to produce H2O2. Storage stability of the milk samples was evaluated through the acidification curves, titratable acidity (TA), total bacterial count (TBC) and coliform counts (CC) at storage times of 0, 6, 12, 18, 24 and 48 hours. The LP activity and the inhibitory effect of activated LPs were evaluated by growing E. coli in pasteurized and boiled camel milk samples as contaminating agent. Results indicated that the W. confusa 22282 activated LPs generally showed significantly (P < 0.05) slower rates of acidification, lactic acid production and lower TBC and CC during the storage time compared to the non-activated sample. The H2O2 producing LAB and exogenous H2O2 activated LPs in pasteurized camel milk significantly reduced the growth of E. coli population compared to non-activated pasteurized milk. Overall, the result of acid production and microbial analysis indicated that the activation of LPs by H2O2 producing LAB (i.e. W. confusa 22282) maintained the storage stability of raw camel milk. Therefore, it can be concluded that the activation of LPs by biological method using H2O2 producing LAB can substitute the chemical activation method of LPs in camel milk.

Current Status of Camel Dairy Processing and Technologies: A Review

Camel milk is unique because it is not easily processed into different dairy products that are common for cow milk. It lacks β-lactoglobulin and has low κ-casein that hinders processing of camel milk into different dairy products. Hence it needs different processing methods and technologies. To this effect attempts were made to manufacture dairy products such as feta cheese, soft brined cheese, and fermented milk from camel milk that had been produced at laboratory level. Camel milk powder has also been manufactures and marketed in Middle East and beyond. Currently there are remarkable progresses in camel dairy technologies that paved the way for production of different camel dairy products at small, medium and large scale. A number of experiments were conducted on camel milk processing technologies from preservation of camel milk using lactoperoxidase system activation using hydrogen peroxide (H2O2) and thiocyanate as well as by H2O2 producing lactic acid bacteria (LAB) to metagenomic characterization of LAB isolated from spontaneously fermented camel milk. Therefore, this review paper is going to present current status of camel dairy technologies in Ethiopia.

Assessment of the Effect of Activated Lactoperoxidase System on Keeping Quality of Raw Cow Milk under Different Climatic Zones of Ethiopia

Assessment of the Effect of Activated Lactoperoxidase System on Keeping Quality of Raw Cow Milk under Different Climatic Zones of Ethiopia

N2 Gas Flushing Limits the Rise of Antibiotic-Resistant Bacteria in Bovine Raw Milk during Cold Storage.

Antibiotic resistance has been noted to be a major and increasing human health issue. Cold storage of raw milk promotes the thriving of psychrotrophic/psychrotolerant bacteria, which are well known for their ability to produce enzymes that are frequently heat stable. However, these bacteria also carry antibiotic resistance (AR) features. In places, where no cold chain facilities are available and despite existing recommendations numerous adulterants, including antibiotics, are added to raw milk. Previously, N2 gas flushing showed real potential for hindering bacterial growth in raw milk at a storage temperature ranging from 6 to 25°C. Here, the ability of N2 gas (N) to tackle antibiotic- resistant bacteria was tested and compared to that of the activated lactoperoxidase system (HT) for three raw milk samples that were stored at 6°C for 7 days. To that end, the mesophiles and psychrotrophs that were resistant to gentamycin (G), ceftazidime (Ce), levofloxacin (L), and trimethoprim-sulfamethoxazole (TS) were enumerated. For the log10 ratio (which is defined as the bacterial counts from a certain condition divided by the counts on the corresponding control), classical Analyses of Variance (ANOVA) was performed, followed by a mean comparison with the Ryan-Einot-Gabriel-Welsch multiple range test (REGWQ). If the storage “time” factor was the major determinant of the recorded effects, cold storage alone or in combination with HT or with N promoted a sample-dependent response in consideration of the AR levels. The efficiency of N in limiting the increase in AR was highest for fresh raw milk and was judged to be equivalent to that of HT for one sample and superior to that of HT for the two other samples; moreover, compared to HT, N seemed to favor a more diverse community at 6°C that was less heavily loaded with antibiotic multi-resistance features. Our results imply that N2 gas flushing could strengthen cold storage of raw milk by tackling the bacterial spoilage potential while simultaneously hindering the increase of bacteria carrying antibiotic resistance/multi-resistance features.

The combined action of the Nisin and Lactoperoxidase system activation on the microbiological quality of raw milk with special emphasis against E.coli O157H7 in milk

Escherichia coli O157:H7 were isolated from 90 bovine and ovine locally produced soft cheese samples and their identification were confirmed based on the cultural, biochemical reactions and serological properties, E.coli O157:H7 isolates obtained by plating on the Chromogenic agars. They were further tested serologically for the presence of both O157 and H7 antigenes using the agglutination test kit. The highest non-significant prevalence (P&gt;0.05) level of E.coli O157:H7 was found in the ewe’s soft cheese samples (37.77%) followed by the cow’s soft cheese samples (31.11%). The antimicrobial potency of the Nisin against the sensitive Lactobacillus strain was lost after (10) minutes of heating at (80°C) while retaining (100%) of its antimicrobial potency after its exposure to the pasteurization time and temperature (63°C/ 30 min.). The highest antimicrobial potency of Nisin was achieved at neutral pH (100%) while 90% and 45% of its potency were retained under acidic (pH=3) and alkaline (pH=9) conditions respectively. Nisin had short bacteriocidal incubation period against the sensitive lactobacillus strain where its antimicrobial potency reduced after 48 hours and lost after 120 hours of refrigeration storage period. E.coli O157:H7 was insensitive to the action of Nisin while stressed E.coli O157:H7 by activation of lactoperoxidase system in pasteurized milk was susceptible to its action and such result gave an indication of the synergistic effect of Nisin with activated lactoperoxidase system.

The combined effect of bacteriocin-producing Lactobacillus rhamnosus GG-ATCC and Lactoperoxidase system activation on microbiological quality of raw milk with special emphasis against E.coli O157:H7 in milk

Isolates of Enterohaemorrhagic E.coli O157:H7 were isolated from 51 and 41 of locally produced bovine and ovine soft cheese samples. Their identification were confirmed based on the biochemical reactions and both the morphological cultural and serological properties. Presumptive E.coli O157:H7 isolates obtained by using the conventional selective plating on the chromogenic agar were tested further for the presence of both O157 and H7 antigenes using the latex agglutination test antisera. The current microbiological studies revealed that 31 (33.70 %) out of 92 bovine and ovine soft cheese samples were positive for E.coli O157:H7. The highest non significant (P&gt;0.05) prevalence level of E.coli O157:H7 was found in the ewe᾿s soft cheese samples (36.59 %) followed by cow᾿s soft cheese samples (31.37 %). Agar well diffusion bioassay method was used for measuring the antibacterial activity of the crude bacteriocin that was produced by Lactobacillus rhamnosus GG-ATCC against Escherichia coli and the closely related sensitive strains such as L.acidophilus LA-K and L.acidophilus ROO52. The crude bacteriocin that was produced by the L.rhamnosus GG-TACC exhibited significantly (P&lt;0.05) the highest antibacterial potency (100 %) against both the closely related strains of lactobacilli and the stressed E.coli O157:H7 by the activation of the LPS. The activation of the natural LPS of inoculated pasteurized milk had significantly (P&lt;0.05) influenced the inactivation degree of the crude bacteriocin against E.coli O157:H7. There was a significant (P&lt;0.05) reduction in the viable counts of stressed E.coli O157:H7 after each exposure time period (6, 24 and 48 hrs.) to the crude bacteriocin at room storage temperature. An overall conclusion on the basis of the current results pointed out that complete elimination of viable bacterial cells was not achieved neither in the stabilized milk (Activation of LPS) nor after subjecting the stabilized milk to the action of the crude bacteriocin produced by L.rhamnosus GG-ATCC at room storage temperature.

Effects of Activated Lactoperoxidase System on Microbiological Quality of Raw Milk

Effects of Activated Lactoperoxidase System on Microbiological Quality of Raw Milk

BEHAVIOR OF AVIRULENT YERSINIA PESTIS IN LIQUID WHOLE EGG AS AFFECTED BY STORAGE TEMPERATURE, ANTIMICROBIALS AND THERMAL PASTEURIZATION

ABSTRACTYersinia spp. are psychrotrophic bacteria capable of growth at temperatures as low as −2C, known to contaminate shell eggs and liquid eggs in the U.S.A. and South America. A study was performed to determine the thermal sensitivity of avirulent Yersinia pestis in liquid whole egg (LWE), evaluate the growth pattern of the bacterium in LWE at temperatures of 4–22C and assess the ability of 10 antimicrobial compounds to inhibit the growth of attenuated Y. pestis in LWE. The estimated decimal reduction values of avirulent Y. pestis in LWE at 54C (D54) were 1.39–1.58 min, and D60 values were 13.8 and 11.4 s by the addition of 0 and 965 IU of nisin (MP Biomedicals, LLC, Solon, OH), respectively. Low molecular weight chitosan (0.5%) and an activated lactoperoxidase system (2.18 U/mL) were ineffective at inhibiting growth of Y. pestis, while 500 IU/mL of nisin inhibited populations by up to 1 log cfu/mL at 4, 10 and 15C when compared with the control. Allyl isothiocyanate, diacetyl, diethyl dicarbonate, ethylenediaminetetraacetic acid, methylparaben, monolaurin and benzoyl peroxide inhibited the growth of attenuated Y. pestis when added at high levels.PRACTICAL APPLICATIONSThe genus Yersinia does not currently pose a problem in pasteurized liquid egg products, although it has been isolated from eggs in the U.S.A. and Argentina. Yersiniae, which are psychrotrophic bacteria, can grow at temperatures as low as −2C; therefore, incidental or intentional contamination of liquid whole egg (LWE) with Yersinia spp. could result in multiplication to high populations, even when stored under refrigeration (ca. 4C). We have shown that avirulent Yersinia pestis is able to multiply to populations of &gt;2, 5 and 8 log cfu/mL in LWE at 4C within 6, 14 and 26 days, respectively. This study provides information that will be helpful in determining thermal and nonthermal means of controlling yersiniae in LWE products.

Relative gene expression in acid-adapted Escherichia coli O157:H7 during lactoperoxidase and lactic acid challenge in Tryptone Soy Broth

Cross-protection of acid-adapted Escherichia coli O157:H7 against inimical stresses is mediated by the glucose-repressed sigma factor RpoS. However, many food systems in which E. coli O157:H7 occurs are complex and contain glucose. This study was aimed at investigating the contribution of acid and lactoperoxidase (LP)-inducible genes to cross-protection of E. coli O157:H7 against LP system and lactic acid (LA) in Tryptone Soy Broth (TSB). Acid-adapted and non-adapted E. coli O157:H7 were challenged to activated LP and LA at pH 4.0 and 5.0 in TSB for 6h at 25°C followed by expression of acid and LP-inducible genes. Acid-adapted E. coli showed cross-protection against activated LP and LA. All the acid-inducible genes tested were repressed at pH 4.0 with or without activated LP system. At pH 7.4, gadA, ompC and ompF were induced in acid-adapted cells. Induction of corA occurred in non-adapted cells but was repressed in acid-adapted cells. Although acid-inducible genes were repressed at pH 4.0, high resistance of acid-adapted cells indicates that expression of acid-inducible genes occurred during acid adaptation and not the actual challenge. Repression of rpoS indicates that RpoS-independent systems contribute to cross-protection in acid-adapted E. coli O157:H7.

Activation of Lactoperoxidase System in Milk by Glucose Oxidase Immobilized in Electrospun Polylactide Microfibers

In this study, glucose oxidase (GOX) was immobilized in polylactide (PLA) fibers that were used to activate the lactoperoxidase (LP) system in milk. The GOX-containing microfibers were electrospun from emulsions prepared by dispersing aqueous GOX in PLA dissolved in a chloroform and N,N-dimethylformamide blend, using sorbitan monopalmitate as an emulsifier. The enzymatic activity of GOX-in-PLA fibers (1100 +/- 400 nm diameter) was more than 19 times higher than that of the GOX-in-PLA membrane formed by direct casting, due to the larger surface area of the electrospun fibers. The activation of LP in model solutions using GOX-in-PLA fibers provided a more sustained generation of antimicrobial OSCN(-) than direct activation using H(2)O(2). Preliminary evaluation on milk samples showed that the electrospun GOX-in-PLA microfibers are capable of activating the naturally present LP system, indicating that they may be promising for active food packaging applications to extend the shelf life of milk.

Calorimetric assessment of microbial growth in milk as affected by different conditions

Milk quality assessment is of paramount importance for dairy facilities. Standard microbiological techniques are time consuming and demand for faster, accurate methods is increasing. On the other hand, inefficient temperature control to handle raw milk, due to lack of appropriate facilities, requires the search for strategies to maximize quality from the microbiological standpoint. Activation of the lactoperoxidase system (LPS) has been established as an easy and safe procedure to attain such results. Good quality milk samples were obtained in sterile containers right after milking. A test temperature of 30 °C was chosen based upon preliminary tests. Raw unpasteurized milk was incubated at 30 °C for 16 h. Microbial growth was assessed at 4 h intervals and the maximum value attained was 4.59×10 6 CFU ml −1 after 8 h. Simultaneously, metabolic heat rate from the same samples was measured under isothermal conditions at 30 °C. A regression equation was obtained to relate microbial growth and heat output, with a coefficient of determination of 0.99. Milk samples were divided in three equal aliquots for (a) pasteurization at 72 °C for 15 s; (b) activation of LPS, and (c) untreated control. LPS activated milk maintained good quality during 8 h, while pasteurized milk showed an initial increase in microbial population after 8 h followed by a decline possibly due to residual activity of its LPS.

PROTEIN PROFILE CHANGES IN ACID ADAPTED LISTERIA MONOCYTOGENES EXHIBITING CROSS-PROTECTION AGAINST AN ACTIVATED LACTOPEROXIDASE SYSTEM IN TRYPTIC SOY BROTH

Foodborne pathogens often tolerate and survive environmental stress conditions including extreme acidity to varying degrees. One possible reason for this survival may be the production of protective stress proteins during acid shock (ASR) and/or tolerance (ATR) responses. The ASR and ATR of Listeria monocytogenes strains V7, V37 and CA in tryptic soy broth without dextrose acidified with lactic acid were studied. Possible cross-protection of acid adapted cells against an activated lactoperoxidase system was also determined. The strains were either directly challenged at pH 4.0 and 3.5 to study their ASR or initially adapted at pH 5.5 for the equivalent of 1 generation before challenging at pH 4. 0 and 3.5 to study their ATR. Adapted and nonadapted cells were challenged at pH 4.5 with or without an activated lactoperoxidase system. In all cases viability was determined by enumeration over a period of 24 or 48 h after challenge and the production of stress proteins analyzed by 2-dimensional gel electrophoresis. While there were some differences in the survival responses for each strain, the acid adapted cells of each strain survived to a greater degree than nonadapted cells at both pH 4.0 (at least 10 fold at 24 h) and pH 3.5 (at least 1000 fold at 6 h) but not at pH 4.5. The acid adapted cells exposed to the lactoperoxidase system survived better (at least 5-fold) than their nonadapted counterparts for all 3 strains at 24 and 48 h. The 2-dimensional gel analysis for all 3 strains showed that the adapted and nonadapted cells underwent a change in their physiology, (at pH 4.0 compared to the control at pH 7.0; at pH 4.5 with the addition of lactoperoxidase system components) in that there was induction as well as repression of several proteins.

Acid Adaptation of Listeria monocytogenes Strains Does Not Offer Cross-Protection against an Activated Lactoperoxidase System

Listeria monocytogenes has been implicated in foodborne illness outbreaks involving several types of cheeses made from acidified milk. Acid shock response (ASR) and acid tolerance response (ATR) could be possible reasons for its survival. The ASR and ATR of three strains of L. monocytogenes (V7, V37, and CA) in skim milk acidified to pH 4.0 and 3.5 with lactic acid and held at 32°C were studied. Studies were also done to determine if acid adaptation of the organism enhanced survival in the presence of an activated lactoperoxidase system. The cells were directly shocked at pH 4.0 and 3.5 in skim milk to study the ASR. To study the ATR, cells were initially adapted in skim milk at a mild pH of 5.5 for the equivalent of one generation before being shocked at pH 4.0 and 3.5 in skim milk. Cells adapted at pH 5.5 in tryptic soy broth without dextrose and nonadapted cells were challenged at pH 4.5 in skim milk with or without an activated lactoperoxidase system. In all cases, viability and pH were measured 24 or 48 h after challenge. In pH 4.0 skim milk, for all three strains, the adapted cell population survived better (0.5 to 1.0 log higher) than that of nonadapted cells for 24 h. In pH 3.5 skim milk, the acid-adapted populations of all three strains were 3 to 4 logs greater than those of nonadapted cells at 6 h. The acid adapted cells of all three strains had survival rates similar to those of the nonadapted cells at pH 4.5 both in the presence and absence of an activated lactoperoxidase system. It was also evident that these strains do not exhibit an adaptive ATR at pH 4.5, although they do at lower pH levels (pH 4.0 and 3.5). Survival due to the ATR was better seen at pH 3.5 than at pH 4.0.

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk.

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4 degrees C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0.21-0.24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8 degrees C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1.87, 1.54 and 1.11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1.99, 2.10 and 1.06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.

Goats' Milk Lactoperoxidase System Against Listeria monocytogenes

A significant decrease in the counts of three Listeria monocytogenes strains was observed in activated lactoperoxidase (LP) system goats' raw milk at refrigeration temperatures during 1–9 d. Inhibitory activity of the LP system on L. monocytogenes depended on temperature, length of incubation, and strain of L. monocytogenes tested. LP system only retarded growth of L. monocytogenes at 20°C in goats' raw milk. Lactoperoxidase was more stable in activated LP system than in control milk at refrigeration temperatures and at 4°C than at 8°C.

Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium.

The relationship of acid adaptation to tolerance of other environmental stresses was examined in Salmonella typhimurium. S. typhimurium was adapted to acid by exposing the cells to mildly acidic conditions (pH 5.8) for one to two cell doublings. Acid-adapted cells were found to have increased tolerance towards various stresses including heat, salt, an activated lactoperoxidase system, and the surface-active agents crystal violet and polymyxin B. Acid adaptation increased cell surface hydrophobicity. Specific outer membrane proteins were induced by acid adaptation, but the lipopolysaccharide component appeared to be unaltered. These results show that acid adaptation alters cellular resistance to a variety of environmental stresses. The mechanism of acid-induced cross-protection involved changes in cell surface properties in addition to the known enhancement of intracellular pH homeostasis.

Poster A6 Chemical and rheological characteristics of manchego cheese made from activated lactoperoxidase system ewes' milk

Poster A6 Chemical and rheological characteristics of manchego cheese made from activated lactoperoxidase system ewes' milk