Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquid foods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. There are many processes that aid in the stability and safety of foods. The combination of different preservation factors, such as nisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation of Listeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selected electric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitization exhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin/ml. A progressive decrease in the population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 1/2 log cycles (U). The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as that exhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism was observed in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocua accomplished by exposure to 10 IU nisin/ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensity of 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin/ml after PEF was 2.8-3.8 U for the selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of the PEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milk containing 1.2 or 37 IU nisin/ml. Inactivation of L. innocua in skim milk containing 37 IU nisin/ml resulted in a decrease in population of 3.7 U.     

Inactivation of high pressure resistant Escherichia coli by lysozyme and nisin under high pressure

We studied the inactivation (by high hydrostatic pressure at 20°C) of Escherichia coli MG1655 and the selected pressure-resistant mutants that were derived previously from this strain, and which are the most pressure-resistant vegetative cells described to date. The natural antimicrobial peptides, lysozyme (50 μg/ml) and nisin (100 IU/ml), enhanced considerably the inactivation of the target bacteria under pressure. However, kinetic inactivation experiments in the presence of these compounds revealed pronounced tailing, which limited the level of inactivation that could be achieved under mild conditions of pressure and temperature. Interrupted pressure treatments enhanced the effectiveness of lysozyme and nisin, allowing a reduction by at least 6 logs of all strains at 400 MPa. A hypothetical mechanism of ‘pressure-promoted uptake’ is proposed to explain E. coli outer membrane permeabilization for lipophilic and cationic peptides like lysozyme and nisin under pressure.     

Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin

Consumer demand for fresh-like products with little or no degradation of nutritional and organoleptic properties has led to the study of new technologies in food preservation. Pulsed electric fields (PEF) is a nonthermal preservation method used to inactivate microorganisms mainly in liquid foods. Microorganisms in the presence of PEF suffer cell membrane damage. Nisin is a natural antimicrobial known to disrupt cell membrane integrity. Thus the combination of PEF and nisin represents a hurdle for the survival of Listeria innocua in liquid whole egg (LWE). L. innocua suspended in LWE was subjected to two different treatments: PEF and PEF followed by exposure to nisin. The selected frequency and pulse duration for PEF was 3.5 Hz and 2 micros, respectively. Electric field intensities of 30, 40 and 50 kV/cm were used. The number of pulses applied to the LWE was 10.6, 21.3 and 32. The highest extent of microbial inactivation with PEF was 3.5 log cycles (U) for an electric field intensity of 50 kV/cm and 32 pulses. Treatment of LWE by PEF was conducted at low temperatures, 36 degrees C being the highest. Exposure of L. innocua to nisin following the PEF treatment exhibited an additive effect on the inactivation of the microorganism. Moreover, a synergistic effect was observed as the electric field intensity, number of pulses and nisin concentration increased. L. innocua exposed to 10 IU nisin/ml after PEF exhibited a decrease in population of 4.1 U for an electric field intensity of 50 kV/cm and 32 pulses. Exposure of L. innocua to 100 IU nisin/ml following PEF resulted in 5.5 U for an electric field intensity of 50 kV/cm and 32 pulses. The model developed for the inactivation of L. innocua by PEF and followed by exposure to nisin proved to be accurate (p = 0.05) when used to model the inactivation of the microorganism by PEF in LWE with 1.2 or 37 IU nisin/ml. The presence of 37 IU nisin/ml in LWE during the PEF treatment for an electric field intensity of 50 kV/cm and 32 pulses resulted in a decrease in the population of L. innocua of 4.4 U.     

Inactivation of Gram-negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system

Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157:H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157:H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination.     

Inactivation of natural microorganisms in litchi juice by high-pressure carbon dioxide combined with mild heat and nisin

The individual and combined effects of high-pressure carbon dioxide (HPCD), mild heat (MH) and nisin (200 ppm) on the inactivation of natural microorganisms, including aerobic bacteria (AB), yeasts and molds (Y&;M), in litchi juice were evaluated. The samples were treated at a pressure of 10 MPa and temperatures of 32, 42 or 52 °C for 5, 10, 15, 20, 25 or 30 min. Temperature played a prominent role in the inactivation of both AB and Y&;M when combined with HPCD, particularly for AB at 52 °C and Y&;M at temperatures ≥42 °C. Nisin increased the susceptibility of AB to the combined treatment of HPCD and MH (HPCD + MH). A reduction of 4.19 log cycles was achieved by HPCD + MH at 52 °C for 15 min, and complete inactivation of AB was obtained by combination of HPCD, MH and nisin (HPCD + MH + nisin). No significant effect of nisin was found on the inactivation of Y&;M.     

Inactivation of Escherichia coli by a combination of nisin,pulsed electric fields,and water activity reduction by sodium chloride

The effect of nisin combined with pulsed electric fields (PEF) and water activity reduction by sodium chloride (NaCl) on the inactivation of E. coli in simulated milk ultrafiltrate media was studied with a Doehlert design and a response surface method. The reduction of water activity from 0.99 to 0.95 by the addition of NaCl (without any other hurdle) did not affect E. coli viability of approximately 10(8) CFU/ml. A reduction in PEF effectiveness occurred when the NaCl concentration was increased because of an increase in conductance, which reduced the pulse decay time. In cells submitted to PEF nisin activity was decreased, probably as a consequence of the nonspecific binding of nisin to cellular debris or the emergence of new binding sites in or from cells. However, the lethal effect due to nisin was reestablished and further improved when water activity was reduced to 0.95. A synergistic effect was evidenced when low-intensity PEF were applied. Decreasing water activity to 0.95 and applying PEF at 5 kV/cm (a nonlethal intensity when no other hurdle is used) with the further addition of nisin (1,200 IU/ml) resulted in a 5-log cycle reduction of the bacterial population.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Quantitative comparison of casein and rapeseed proteolysis by pancreatin

The problem of quantitative comparison of kinetic curves was solved for casein and rapeseed pancreatin hydrolysis in a membrane reactor, which ensured the measurement of proteolysis kinetics for the products with a molecular weight of less than 1000. Coordinates were derived which provided good linearization of kinetic curves and the determination of relative rate constants irrespective of reagent concentrations. E₀/S₀ ratio and time intervals of kinetic measurements. When the relative rate constants of the release of the individual amino acid residues in the low-weight proteolysis products were compared, trypsin-dependent constants (for Lys and Arg residues) were found to be two times less for rapeseed than for casein, and chymotrypsin-dependent constants (for Tyr and Phe residues) were approximately 1.3 times higher for rapeseed than for casein. Statistical analysis demonstrated that the distribution of constants was narrower for rapeseed than for casein. Differences between target (Arg, Lys, Tyr and Phe) and non-target constants of release in the form of peptides and free amino acids, or in the form of free amino acids only, were attributed to the differences in the peptide bond masking for casein and rapeseed proteins. Computer simulation of proteolysis kinetics was performed by PROTEOLYSIS program package to confirm the dependence of rate constant distribution on the state of masking.     

Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon

Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C. Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E. coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days. The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells. The pathogens were gradually inactivated by the acidic pH of apple juice. Nisin and cinnamon greatly contributed to the inactivation. The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E. coli O157:H7. Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature. The TAL method was as effective as TSA in recovering injured cells of the pathogens. The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E. coli O157:H7 in apple juice and so enhances the safety of the product.     

Inactivation of Listeria innocua and Escherichia coli in carrot juice by combining high pressure processing,nisin, and mild thermal treatments

This study explored the effectiveness of high pressure (200–500 MPa) alone or in combination with mild thermal treatments (35 and 50 °C) and nisin (25 and 50-ppm) on the inactivation of L. innocua and E. coli in carrot juice. Processing at 500 MPa at 20 °C for 2 min without nisin resulted in 4- and 5-log CFU/mL reduction of L. innocua and E. coli, respectively while incorporating 25-ppm nisin at same pressure and temperature rendered 7-log CFU/mL reduction. There was synergism between high pressure, nisin, and heat in all treatments to inactivate both microorganisms. After a 28-d of refrigerated storage, total plate counts were <2-log CFU/mL in carrot juice treated with combination of 300 MPa and 25-ppm nisin at 35 °C. All combinations resulted in less intense use of pressure, i.e. more energy efficient, cost effective processes while attaining high quality juices. The results of this study suggest that by using selected combinations of high pressure, nisin and mild temperatures, safe, clean-label, high-quality juices can be produced.Industrial relevanceThe results from this study show a synergistic effect on the inactivation of L. innocua and E. coli in carrot juice from the combined application of HPP, nisin, and mild temperatures. By replacing the use of HPP alone by these combinations will allow the use of reduced pressures over shorter period of times to process low-acid juices, lowering energy requirements and increasing throughput. This study will aid the beverage processing industry in the development of clean label juice products with fresh-like quality attributes and using considerable less energy to conventional processing.     

Analysis of nisin A,nisin Z and their degradation products by LCMS/MS

The peptides nisin A and nisin Z belong to type-A lantibiotics applied as preservatives in cheese production. The present study optimised and validated a liquid chromatography–tandem mass spectrometry (LCMS/MS) method for the analysis of nisin A in cheese. Since nisin A was not detectable in nisin-containing commercial cheese samples, an additional LCMS/MS method for the quantification of nisin Z was developed and validated. Quantification was performed by external calibration and standard addition. The latter method provided a non-significantly higher recovery rate for the tested cheese matrix. During the production of processed cheese, nisin A and nisin Z undergo significant degradation. Six degradation products of nisin A or nisin Z, respectively, were detected and assigned to nisin A/Z + H₂O, nisin A/Z^1–32, and nisin A/Z^1–32 + H₂O. In two out of eight commercial processed cheese samples, 1.6, resp. 1.7 mg nisin Z/kg cheese was measured, whereas nisin A was not detectable in any of the samples.     

固定化细胞发酵生产乳链菌肽工艺研究

乳链菌肽是由乳酸乳球菌代谢产生的细菌素,它对许多革兰氏阳性菌(如葡萄球菌、链球菌、微球菌、李斯特氏菌和乳杆菌等病原菌和腐败菌)有强烈的抑杀作用,又因为在消化道中能很快的被α-胰凝乳蛋白酶分解,对人体无不良反应。是一种天然、安全的生物防腐剂,被广泛应用于食品工业中。利用固定化细胞技术发酵生产乳链菌肽能够简化工业生产的流程,优化乳链菌肽的生产工艺。本文以乳酸乳球菌为菌种,活化培养后固定于海藻酸钠、琼脂、明胶-戊二醛三种载体上。分别选取不同的胶珠直径、发酵时间、发酵温度、载体浓度、接种量进行培养实验,检验不同工艺条件对乳链菌肽效价的影响,选取最佳发酵工艺条件。研究结果表明最佳工艺条件为:海藻酸钠溶液的浓度为2.0%,胶珠直径为2².5mm,接种量为5%,37℃发酵24h。     

胰酶酶解珍珠贝分离蛋白制备低苯丙氨酸寡肽制品

苯丙酮酸尿症(PKU)是一种常见的氨基酸代谢性疾病,低苯丙氨酸饮食对于PKU患者非常重要。本研究以珍珠产业副产物马氏珠母贝肉为原料,提取分离蛋白后,采用胰酶酶解、超滤分级及活性炭吸附分离技术制备低苯丙氨酸寡肽营养功能基料。研究结果显示:随着酶解时间的进行,酶解液中<5 ku的寡肽含量呈现先增加后减少趋势(p<0.05),酶解1 h时,酶解液中所含的寡肽含量最多;经过超滤分级后,>5 ku和<300 u的酶解液中仍存在一些寡肽残留,其中>5 ku组分中寡肽含量为10.43%,<300 u组分含量为24.52%,而300 u~5 ku组分中寡肽含量达到了68.38%;<300 u组分的苯丙氨酸含量最高达到9.52%,活性炭对芳香族氨基酸的吸附量均高于其他氨基酸,300 u~5 ku组分经过活性炭吸附后得到1.29%的低苯丙氨酸寡肽溶液。可以以此为基料开发辅助治疗苯丙酮尿症、吸收率高的低苯丙氨酸寡肽营养功能制品。      

乳链菌肽对搅拌型酸奶后发酵的抑制效果研究

为了延长酸奶的货架期，其后发酵必须得到有效地控制。通过测定添加不同量尼萨普林的酸奶在货架期内酸度、pH值等指标，得知尼萨普林对酸奶（4℃及25℃贮藏）的后发酵具有显著的抑制作用，并确定尼萨普林的最佳用量为0．15g/kg。     

胰酶提升小麦蛋白酶解产物乳化活性工艺及其功能特性研究

以乳化活力为指标,系统地研究了胰酶水解小麦蛋白的工艺条件,酶解产物的抗氧化活性、分子量分布、氨基酸组成等功能特性。结果表明,最佳酶解条件为:酶浓度E/S为1%、温度45℃、pH7.5,乳化活力指数达到35.38m2/g,此时水解度为4.73%,显著提升了小麦蛋白的乳化活性。最佳工艺条件下的小麦蛋白酶解液具有一定的DPPH自由基清除能力,但显著低于VC;首次发现了酶解液具有较强的金属螯合能力。酶解小麦蛋白产物中多肽分子量主要分布在200~1000u,占65.99%;酶解产物中共检测出了15种氨基酸,谷氨酸含量最高,达30.48%;肽类可能是酶解产物具备化活性、抗氧化活性等功能特性的重要贡献者。      

Synergistic inhibition of Listeria monocytogenes by nisin and garlic extract

Minimum Inhibitory Concentrations (MICs) were determined for nisin and an aqueous garlic extract (AGE) for six strains of Listeria monocytogenes, including a nisin-resistant mutant. When used in combination in broth a synergistic effect was observed between nisin and AGE in all strains. Increasing the pH reduced the activity of both nisin and garlic. During storage of the aqueous garlic extract at 4°C and at 20°C for 14 days, the MIC increased over the first 8 days but remained stable thereafter, indicating at least two antimicrobial components, one of which was relatively stable. Aqueous garlic extract was not appreciably bactericidal although there was a positive interaction with nisin in terms of its bactericidal effect in broth at 4°C. The effect of combining nisin and garlic in a food system, hummus, was also studied. Sub-MIC combinations of AGE and nisin were effective at preventing listerial growth and did enhance the slight bactericidal effect of nisin. The results indicate that combined use of nisin with garlic could help overcome problems of nisin resistance in Gram-positive organisms. Such interactions could also be a significant factor in traditional lactic fermented foods where garlic is an ingredient.     

以乳清为原料采用混菌发酵技术促进Nisin生产的研究

采用了一种不用外加碱或移除乳酸来达到控制发酵液中乳酸的新技术,即采用乳酸菌和酵母菌的混和发酵来控制pH值以促进乳酸链球菌素(nisin)的生产。其原理是乳酸菌在发酵生产nisin的过程中所产生的乳酸可以被酵母菌利用,从而达到控制发酵液pH值的目的;同时,混菌培养选用的酵母菌不能利用乳清培养基中的乳糖,从而避免了2株菌之间对碳源的竞争,创造了一个互利共生的微生态环境,从而达到促进nisin生产的目的。实验表明,乳酸根的积累对nisin发酵生产具有抑制作用;混合发酵的酵母菌利用了发酵液中的乳酸,促进了乳酸菌生长和nisin生产;酵母菌较乳酸菌提前3h接入到发酵培养基中时,能更好地控制pH值;最佳的接种比例为乳酸菌3%,酵母菌5%。