Inactivation of food spoilage bacteria by high pressure processing: Evaluation with conventional media and PCR–DGGE analysis

Microbial diversity and dynamic changes of sliced vacuum-packed cooked ham during refrigerated storage (0–90 days) after high pressure processing (400 MPa at 22 °C for 10 min) was investigated by using culture-dependent and culture-independent approaches. Isolation of genome DNA and total RNA directly from meat samples, followed by PCR–denaturing gradient gel electrophoresis (DGGE) and RT-PCR–DGGE on 16S rDNA V3 region, was performed to describe the structure of the bacterial community and active species in pressurized sliced cooked ham. The DGGE profile showed that most spoilage bacteria including Lactococcus garvieae, Weissella cibaria, Lactobacillus sakei, Lactobacillus curvatus, Weissella paramesenteroides, Leuconostoc carnosum and Lactococcus lactis subsp. lactis were completely inactivated after high pressure processing (HPP), whereas Weissella viridescens and Weissella minor survived HPP and induced the final spoilage. The microbial diversity of HPP samples during the whole refrigerated storage period was extremely simple. Our results clearly indicated that HPP was an efficient method for avoiding the growth of the major spoilage bacteria and could be used to prolong the shelf-life of sliced vacuum-packed cooked ham.     

Selective effect of the product type and the packaging conditions on the species of lactic acid bacteria dominating the spoilage microbial association of cooked meats at 4°C

The product type was shown to strongly affect the growth rate and the composition of the spoilage lactic flora during refrigerated (4°C) storage of cooked, cured meats, sharing their processing plant environment, day of production and film packaging conditions. Growth of lactic acid bacteria (LAB) under vacuum was more prolific on the product in the order: ham>turkey breast fillet>smoked pork loin>pariza>mortadella>bacon, and ham>frankfurters, manufactured in two industrial meat plants A and B, respectively. The Lactobacillus sakei/curvatus group prevailed in all products, except the non-smoked, boiled whole-meats, i.e. cooked ham and turkey breast fillet, where Leuconostoc mesenteroides subsp.mesenteroides predominated. Lactobacillus sakei was by far the most prevalent species in smoked whole-meats, i.e. pork loin and bacon. Emulsion sausages, i.e. pariza, mortadella and frankfurters, contained a more diverse lactic flora.Leuconostoc carnosum and Lc. citreum occurred in boiled, whole-meats and emulsion sausages, respectively.Weissella viridescens was isolated from smoked meat products only. A very good correlation between the LAB growth and types and important intrinsic factors, such as the product pH, moisture, salt (brine) concentration and cooking method could be observed. When ham and frankfurters from plant B were stored in air, yeasts and mainly Brochothrix thermosphacta became important members of the spoilage association. Growth of LAB was faster in air. The presence of oxygen resulted in a replacement ofLc. mesenteroides subsp. mesenteroides by other Leuconostoc spp. in ham, and in a shift of the spoilage flora from homo- to heterofermentative LAB species in frankfurters.     

Effect of high pressure treatment on microbial populations of sliced vacuum-packed cooked ham

In this study, culture-dependent and culture-independent approaches were used to reveal the microbial diversity and dynamic changes occurring in sliced vacuum-packed cooked ham after high pressure processing (HPP, 400MPa or 600MPa for 10min at 22°C) during refrigerated storage over 90days. Direct extraction of genome DNA and total RNA from meat samples, followed by PCR-denaturing gradient gel electrophoresis (DGGE) and RT-PCR-DGGE on 16S rDNA V3 region, was performed to define the structure of the bacterial populations and active species in pressurized cooked ham. Results showed that HPP affected differently the various species detected. The predominant spoilage organisms of cooked ham, such as Lactobacillus sakei and Lactobacillus curvatus, were found to be very sensitive to pressure as they were unable to be detected in HPP samples at any time during refrigerated storage. Weissella viridescens and Leuconostoc mesenteroides survived HPP at 600MPa for 10min at 22°C and were responsible for the final spoilage. An RNA-based DGGE approach clearly has potential for the analysis of active species that have survived in pressurized cooked ham. High pressure processing at 400 or 600MPa for 10min at room temperature (22°C) has a powerful inhibitory effect on the major spoilage bacteria of sliced vacuum-packed cooked ham. High pressure treatment may lead to reduced microbial diversity and improve the products' safety.     

Protective cultures inhibit growth of Listeria monocytogenes and Escherichia coli O157:H7 in cooked, sliced, vacuum- and gas-packaged meat

Contamination of cooked meat products with Listeria monocytogenes poses a constant threat to the meat industry. The aim of this study was therefore to investigate the use of indigenous lactic acid bacteria (LAB) as protective cultures in cooked meat products. Cooked, sliced, vacuum- or gas-packaged ham and servelat sausage from nine meat factories in Norway were inoculated with 10(3) cfu/g of a mixture of three rifampicin resistant (rif-mutant) strains of L. monocytogenes and stored at 8 degrees C for four weeks. Growth of L. monocytogenes and indigenous lactic acid flora was followed throughout the storage period. LAB were isolated from samples where L. monocytogenes failed to grow. Five different strains growing well at 3 degrees C. pH 6.2, with 3% NaCl, and producing moderate amounts of acid were selected for challenge experiments with the rif-resistant strains of L. monocytogenes. a nalidixic acid/streptomycin sulphate-resistant strain of Escherichia coli O157:H7 and a mixture of three rif-resistant strains of Yersinia enterocolitica O:3. All five LAB strains inhibited growth of both L. monocytogenes and E. coli O157:H7. No inhibition of Y. enterocolitica O:3 was observed. A professional taste panel evaluated cooked, sliced, vacuum-packaged ham inoculated with each of the five test strains after storage for 21 days at 8 degrees C. All samples had acceptable sensory properties. The five LAB strains hybridised to a 23S rRNA oligonucleotide probe specific for Lactobacillus sakei. These indigenous LAB may be used as protective cultures to inhibit growth of L. monocytogenes and E. coli O157:H7 in cooked meat products.     

牛肉火腿切片的腐败微生物鉴定及贮藏过程中的品质变化

对牛肉火腿切片的腐败微生物进行了鉴定。结果表明,腐败产品中的细菌主要是乳杆菌属兼性异型发酵菌——干酪乳杆菌(Lactobacillus casei)。干酪乳杆菌发酵产酸、产气是牛肉火腿切片胀袋、出水的根本原因。并研究了贮藏过程中不同包装、温度、光照条件对品质变化的影响。光照对真空包装的牛肉火腿切片的脂肪氧化及色泽变化影响都较小,但对气调包装的a值影响较大,造成褪色明显。保持低温环境对于抑制乳杆菌的生长繁殖,延长产品货架期具有重要作用。     

Competitive inhibition of Listeria monocytogenes in ready-to-eat meat products by lactic acid bacteria

Forty-nine strains of lactic acid bacteria (LAB), isolated from commercially available ready-to-eat (RTE) meat products, were screened for their ability to inhibit the growth of Listeria monocytogenes at refrigeration (5 degrees C) temperatures on agar spot tests. The three most inhibitory strains were identified as Pediococcus acidilactici, Lactobacillus casei, and Lactobacillus paracasei by 16S rDNA sequence analysis. Their antilisterial activity was quantified in associative cultures in deMan Rogosa Sharpe (MRS) broth at 5 degrees C for 28 days, resulting in a pathogen reduction of 3.5 log10 cycles compared to its initial level. A combined culture of these strains was added to frankfurters and cooked ham coinoculated with L. monocytogenes, vacuum packaged, and stored at 5 degrees C for 28 days. Bacteriostatic activity was observed in cooked ham, whereas bactericidal activity was observed in frankfurters. Numbers of L. monocytogenes were 4.2 to 4.7 log10 and 2.6 log10 cycles lower than controls in frankfurters and cooked ham, respectively, after the 28-day refrigerated storage. In all cases, numbers of LAB increased by only 1 log10 cycle. The strain identified as P. acidilactici was possibly a bacteriocin producer, whereas the antilisterial activity of the other two strains was due to the production of organic acids. There was no significant difference (P > 0.05) in the antilisterial activity detected in frankfurters whether the LAB strains were used individually or as combined cultures. Further studies over a 56-day period indicated no impact on the quality of the product. This method represents a potential antilisterial intervention in RTE meats, because it inhibited the growth of the pathogen at refrigeration temperatures without causing sensory changes.     

Influence of pasteurization,brining conditions and production environment on the microbiota of artisan Gouda-type cheeses

To monitor the effect of the indigenous milk microbiota and of technological and environmental parameters on the microbiota established in ripened cheese, the diversity and dynamics of the predominant microbial communities in artisan Gouda-type cheeses produced under different conditions was studied. A total of 22 cheese types differing in milk source, milk treatment, production environment and brining conditions were analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) using total DNA extracts as well as DNA extracted from culturable fractions. Through band position analysis and band sequencing, the majority of DGGE bands could be attributed to lactic acid bacteria (LAB), although a few bands also belonged to staphylococci and gamma-Proteobacteria. Aided by principal component analysis (PCA) and multivariate analysis of variance (MANOVA), cheeses produced at different locations could clearly be differentiated. The same approach also allowed to distinguish raw and pasteurized milk cheeses, the former showing a more diverse microbiota in terms of a higher species richness and number of DGGE bands. No substantial differences were found between cheeses brined at two different locations. In conclusion, the combined PCR-DGGE approach relying on both total DNA extracts and culturable fractions proved its value for analyzing the effect of technological and environmental parameters on the diversity and dynamics of the microbiota in Gouda-type cheeses.     

A culture-dependent and -independent approach for the identification of lactic acid bacteria associated with the production of nem chua,a Vietnamese fermented meat product

In this study, the diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented meat, was described using a combination of culture-dependent and culture-independent methods. A total of two hundred seventy-three LAB isolates were subjected to a polyphasic identification approach combining (GTG)₅-PCR fingerprinting and phenylalanyl-tRNA synthase α subunit (pheS) and RNA polymerase α subunit (rpoA) gene sequence analysis. LAB associated with nem chua were identified as Lactobacillus pentosus (21%), Lactobacillus plantarum (29.7%), Lactobacillus brevis (5%), Lactobacillus paracasei (0.4%), Lactobacillus fermentum (0.7%), Lactobacillus acidipiscis (0.4%), Lactobacillus farciminis (23%), Lactobacillus rossiae (0.4%), Lactobacillus fuchuensis (0.7%), Lactobacillus namurensis (0.4%), Lactococcus lactis (0.4%), Leuconostoc citreum (9.5%), Leuconostoc fallax (1%), Pediococcus acidilactici (1%), Pediococcus pentosaceus (4%), Pediococcus stilesii (1%), Weissella cibaria (0.7%) and Weissella paramesenteroides (0.7%). Furthermore, PCR-DGGE was also applied as a culture-independent method in this study. Results indicated the presence of species of which no isolates were recovered, i.e. Lactobacillus helveticus/crispatus, Lactococcus garvieae and Vagococcus sp. Conversely, not all isolated bacteria were detected by PCR-DGGE. Principal component and discriminant analysis disclosed correlations between the different production locations and certain isolated LAB species and strains and/or DGGE bands suggesting possible influences of locally prevailing production practices on the nem chua LAB microbiota.     

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork.     

Nondestructive Monitoring of Oxygen Profiles in Packaged Foods Using Phase-Fluorimetric Oxygen Sensor

ABSTRACT: Nondestructive quantitation of oxygen in different types of packaged foods was performed using disposable phosphorescent oxygen sensor inserts placed in every individual sample and a fiber-optic phosphorescent phase detector. Oxygen levels and their changes over storage time are presented for vacuum-packed raw and cooked meat, smoked fish, and MAP sliced ham and bread. Damage to vacuum-packages was simulated by slitting the package film and monitoring the sensor response at different locations from the site of damage. The performance of the optical oxygen sensor in packaged foods was evaluated, and its usefulness for food research and industrial applications discussed.     

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.     

不同贮藏温度西式火腿切片品质变化规律研究

为研究市场上低温火腿切片的质量和安全性,对不同贮藏温度的真空包装火腿切片的品质变化进行动态跟踪,分析产品在0～4、7～11℃条件下,其感官品质、菌落总数、大肠菌群、色泽、pH值、水分含量、保水性和质构特性的动态变化。结果表明：两种产品品质变化的规律大致相同。0～4℃贮藏比7～11℃产品的菌落总数增长相对缓慢,pH值、水分含量,保水性和质构特性的变化更加稳定。低温更有利于保持产品品质的稳定性和安全性,延长肉品的货架期。     

Impact of slicing hygiene upon shelf life and distribution of spoilage bacteria in vacuum packaged cured meats

A model for studying spoilage in sliced cooked delicatessen meats using conventional plating procedures was characterized by following the transfer of adventitious lactic acid bacteria (LAB), thermotolerant LAB and pediococci from a dry fermented sausage to cooked ham and bologna during a simultaneous slicing procedure. The major group of organisms present in these products was LAB. Each of the three groups of organisms was transferred to the cooked meats, but during storage under vacuum at 7°C, only the adventitious lactobacilli from the sausage appeared to contribute to cooked meat spoilage. Pediococci declined within 28 days of co-slicing, thermotolerant LAB grew slowly and never reached levels of >5 log₁₀cfu cm⁻², but transferred adventitious LAB from the sausage grew and reached peak populations 4 weeks earlier in the co-sliced cooked meats than in controls. During storage at 7°C co-sliced cooked meats spoiled, as assessed by purge (liquid) and ropy slime formation, 2–4 weeks faster than the separately-sliced and vacuum-packaged products. The distribution of organisms on package slices differed. In control cooked products numbers of bacteria present on center package slices were lower than on outside slices. Bacterial numbers were greatest at the meat surface–packaging film interface. In co-sliced cooked products organisms were evenly distributed throughout slices in the packages. Based on findings from use of the co-slice model, in vacuum packages of sliced cured meats where >10⁵cfu bacteria cm₋₂are equally distributed throughout package slices, poor slicing hygiene is the cause. Commercially-sliced bologna was repackaged with allyl isothiocyanate (AIT) to test its ability to delay bacterial growth at the meat surface. The preservative effects of AIT were tested under vacuum or when backflushed with CO₂. CO₂alone was more effective than AIT in delaying LAB growth but neither treatment was particularly effective.     

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork.     

Industrial application of an antilisterial strain of Lactobacillus sakei as a protective culture and its effect on the sensory acceptability of cooked, sliced, vacuum-packaged meats.

The application of a protective lactic acid bacterium (LAB) during the commercial production of cooked meat products is described. The LAB, a strain of Lactobacillus sakei, was previously isolated from cooked ham and inhibited growth of Listeria monocytogenes and Escherichia coli O157:H7 in this product. L. sakei was applied to the cooked products at a concentration of 10(5)-10(6) cfu/g immediately before slicing and vacuum-packaging using a hand-operated spraying bottle. The LAB strain inhibited growth of 10(3) cfu/g of a cocktail of three rifampicin resistant mutant L. monocytogenes strains both at 8 degrees C and 4 degrees C. Consumer acceptance tests of cooked ham and of servelat sausage, a Norwegian non-fermented cooked meat sausage, showed that control and inoculated products were equally acceptable. The products were still acceptable after storage for 28 days at 4 degrees C and, after opening the packages, for a further 5 days at 4 degrees C. The findings presented here confirm that the L. sakei strain is suitable for use as a protective culture and may technically easily be implemented in the commercial production of cooked meat products.     

Inhibition of potential food poisoning microorganisms by sorbic acid in cooked, uncured, vacuum packaged turkey products

Vacuum packaged, oven-roasted turkey breasts and sliced turkey breast luncheon meat were prepared with and without potassium sorbate or sorbic acid. Control and treated products were inoculated with one of the following organisms: Salmonella, Staphylococcus aureus , or enteropathogenic Escherichia coli. The samples were vacuum packed and stored at 15°C for 10 days. The addition of 0.25% sorbate to the breasts and 0.12% sorbic acid in the slices provided excellent protection against the growth of Salmonella, E. coli , and S. aureus in uncured, cooked, vacuum packaged turkey.     

Effect of Dietary Supplementation with α-Tocopheryl Acetate on the Stability of Reformed and Restructured Low Nitrite Cured Turkey Products

The objective of this study was to investigate the effects of sodium (Na) nitrite reduction on the oxidative and colour stability of reformed and restructured cured cooked turkey products manufactured from meat containing high and low levels of dietary α-tocopheryl acetate. Turkeys were randomly assigned to either a control group, fed a basal α-tocopheryl acetate diet (20mg/kg feed), or a treatment group fed a supplemented α-tocopheryl acetate diet (600mg/kg feed). Diets were fed ad libitum from day 1 until slaughter on day 147. Breast meat from control and treatment groups was used to manufacture cured reformed cooked turkey ham and cured restructured cooked turkey patties. Residual levels of 60 and 120mg Na nitrite/kg of meat were used. Turkey products were packaged in either overwrap or vacuum packaging and stored under refrigerated (4°C) illuminated display for 10 days. Results showed that dietary supplementation with α-tocopheryl significantly (p<0·05) improved the oxidative and colour stability of all low nitrite products produced when compared to non-supplemented controls.     

Study of the Lactobacillus sakei protective effect towards spoilage bacteria in vacuum packed cooked ham analyzed by PCR-DGGE

The effectiveness of Lactobacillus sakei B-2 inoculated as a protective culture on the inhibition of spoilage bacteria on sliced vacuum packed cooked ham was investigated by using culture-dependent and -independent approaches. Total microbial DNA was directly extracted from both control and treatment samples, and subjected to a nested PCR protocol, PCR–DGGE analysis was used to identify and monitor the dynamic changes in the microbial population, followed by partial 16S rDNA sequencing. The DGGE profile demonstrated that the protective culture effectively suppressed growth of predominant spoilage bacteria L. sakei, Lactobacillus curvatus and Leuconostoc mesenteroides in cooked ham during storage at 4 °C, however, growth of uncultured Leuconostoc was not inhibited. The shelf-life of this product inoculated with L. sakei B-2, at levels of 5.91 ± 0.04 log₁₀ CFU g⁻¹ was 35 days, compared to 15 days of control samples, when the ham was stored at 4 °C.     

Microflora of Feta cheese from four Greek manufacturers

The components of the microflora of four Feta cheeses, produced by different Greek manufacturers, were determined by culture dependent and independent techniques. Isolates from microbiological media were first grouped by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and then representatives of each DGGE group were sequenced for identification purposes. DNA and RNA, extracted directly from the cheese, were subjected to PCR-DGGE. Moreover, Feta cheeses were subjected to FISH analysis in order to identify viable bacterial populations. The microbial ecology, as represented by the Lactic Acid Bacteria (LAB) and yeast populations, was different for the four cheeses. The main LAB species isolated were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coryniformis and Lactobacillus fermentum. However, some inconsistencies were observed between the results obtained with the culture dependent and the culture independent approach. In the case of the yeasts, the results obtained by PCR-DGGE compared very well with those obtained by the conventional microbiological analysis and the main species found were Kluyveromyces lactis, Pichia fermentans and C. zeylanoides. FISH analysis highlighted viable but not culturable populations of Streptococcus thermophilus and Lactococcus spp. RAPD-PCR performed on the L. plantarum isolates revealed a cheese specific distribution and a temperature dependent clustering.     

Growth ofListeria monocytogeneson sliced cooked meat products

During the shelf life (4–6 weeks) of artificially contaminated sliced cooked meat products such as luncheon meat, ham and chicken breast, the growth ofListeria monocytogenesunder vacuum was similar to the growth under modified atmosphere (30% CO₂/70% N₂) packaged products. The presence of competitors (lactobacilli), even in concentrations 100 times those ofL. monocytogenes, only slightly inhibited growth of this pathogen. At the end of the shelf life levels were still 10⁷cfug⁻¹. Due to the lower initial contamination, levels in naturally contaminated products were about 10⁴cfug⁻¹. To prevent outgrowth ofL. monocytogenesto such high levels it is necessary to prevent recontamination during slicing and packaging, and to shorten the rather long shelf life of these products. Due to the low pH of fermented sausage (saveloy) and (raw) Coburger ham the numbers ofL. monocytogenesdecreased below the detection level.