Meat Starter Cultures Research Articles

Meat Starter Culture Reduces Aspergillus parasiticus Production of Aflatoxins on Meat-Based and Salami Model Media.

There is great concern about the risk posed by the consumption of food contaminated with aflatoxins (AF), produced mostly by Aspergillus strains, that can also be found in dry-fermented meat products (DFMPs). The aim of this study was to investigate the inhibitory effect of meat starter culture (SC), frequently used for fermentation in the meat industry, on A. parasiticus growth and the production of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and sterigmatocystin (STE) on different meat-based (CMA) and salami model (SM-G) media. Incubation was carried out under optimal conditions for fungal growth and under typical conditions for ripening of DFMPs for 21 days. Reversed-phase UPLC-MS/MS analysis was performed to determine mycotoxin production. SC reduced A. parasiticus growth more on CMA than on SM-G media. AFB1 formation was inhibited on both types of SC-containing media, although SC generally had a stronger inhibitory effect on AFB1 production on CMA than on SM-G. AFB1 and AFB2 were produced on CMA, while AFB1 dominated in SM-G, AFG1, and AFG2 were not detected in any media. The results show that SC inhibited AFB1 formation of A. parasiticus on SM-G media after 21 days of incubation under typical conditions for the production of DFMPs. These results indicate the necessity to investigate AF on natural matrices in an environment that is as similar as possible to real conditions in the production of DFMPs.

Role of Curing Agents in the Adaptive Response of the Bioprotective Latilactobacillus curvatus CRL 705 from a Physiologic and Proteomic Perspective

During meat processing, lactic acid bacteria (LAB) have to competitively adapt to the hostile environment produced by curing additives (CA). The objective of this study was to investigate the ability of Latilactobacillus curvatus CRL 705, a bioprotective strain of meat origin, to adapt to CA. A physiological and proteomic approach was performed. CRL 705 was grown in a chemically defined medium (CDM) containing specific concentrations of CA (NaCl, nitrite, sucrose, and ascorbic acid). The results showed minor differences in growth kinetics in the presence of CA. Glucose consumption, present in the CDM, and production of lactic acid and bacteriocins were not significantly affected. Proteomic analyses indicated that most of the identified proteins (36 out of 39) mainly related to carbohydrate metabolism (18%), posttranslational modifications (15.6%), energy production and conversion (11.1%), translation (11.1%), and nucleotide metabolism (8.9%) were underexpressed. In response to the studied CA, CRL 705 slowed down its general metabolism, achieving slight changes in physiological and proteomic parameters. The observed performance is another characteristic that extends the well-known competitive profile of CRL 705 as a meat starter and bioprotective culture. This is the first report dealing with the impact of CA on LAB proteomics.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages.

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

Alternative culture medium design for biomass production of autochthonous meat starter Latilactobacillus sakei sp. sakei ACU-2

The autochthonous strain Latilactobacillus sakei sp. sakei ACU-2 was selected as a meat starter culture for dry sausage production. Transferring this strain from laboratory scale to industry requires an increase in biomass production, while lowering process costs. In this study, a combination of techniques was applied in order to optimize the culture medium composition to enhance biomass production of L. sakei ACU-2. One variable at a time experiments, Plackett–Burman design, and mixture design were performed to fulfill the strain nutritional requirements. Eventually, the optimized formulation contained 19.46 g/L yeast extract; 8.28 g/L whey protein concentrate; 2.26 g/L soy peptone; 30 g/L cerelose; 1 g/L Tween 80; 5 g/L sodium acetate; 0.2 g/L magnesium sulfate and 0.05 g/L manganese sulfate. When L. sakei ACU-2 was cultivated in a bioreactor using the alternative medium, an enhancement of 75.5% of biomass production was achieved, in comparison to its growth in the commercial de Man, Rogosa, and Sharpe medium. Furthermore, a reduction of 62–86% of the cost was also attained. These results support a promising large-scale application of the designed medium for high biomass yields of the starter culture at minor costs.

Proteomic response strategies of Pediococcus pentosaceus R1 isolated from Harbin dry sausages to oxidative stress

The tolerance strategies of Pediococcus pentosaceus R1 to sublethal dose of H2O2 induced-oxidative stress were investigated with proteomic approaches. The results revealed that a total of 117 proteins were identified as differentially expressed proteins. Eight antioxidant enzymes were upregulated expression to defence the oxidative stress, especially manganese catalase. Besides, 14 proteins participating in carbohydrate and energy metabolism, were significantly upregulated expression (P < 0.05), in which six proteins involved in the two major pathways, pentose phosphate pathway and pyruvate metabolism. Furthermore, the results showed that the expression of four proteins related to the repair of oxidatively damaged proteins was upregulated (P < 0.05); and the upregulated expression was also found in five ATP-binding cassette transporter-related proteins, which involved in defence oxidative stress in P. pentosaceus R1 by regulating the transmembrane transport of many substrates (P < 0.05). Conversely, the expression of 13 proteins related to nucleotide biosynthesis, and four proteins participating in Fe–S cluster assembly, was significantly downregulated (P < 0.05). This work will facilitate the understanding of the antioxidative mechanisms in P. pentosaceus R1 and will provide valuable information for further engineering oxidation-tolerant strains as meat starter cultures.

Genome Sequencing of Kocuria varians Strain 80, Isolated from Starter Culture for Dry Sausage.

The genome of the meat starter culture strain Kocuria varians 80 was sequenced and assembled into a single 2.82-Mb chromosome. Genome sequence comparison of K. varians strain 80 and previously sequenced strains revealed predicted proteomic differences that can impact its technological properties.

Detection of Biogenic Amines and Tyramine-Producing Bacteria in Fermented Sausages from Switzerland

Fermented foods can cause human illness because of the unhealthy effect of biogenic amines (BAs) that accumulate by decarboxylation of free amino acids. Salami-type fermented sausages can contain BAs, but which bacteria and environmental factors contribute to BA production is not clear. Sixty-two sausages purchased from Swiss markets were evaluated for decarboxylating bacterial strains and concentrations of the BAs cadaverine, histamine, putrescine, and tyramine. Based on the size and number of employees of the meat processing plants, sausages were separated into two groups: artisanal and industrial. Concentrations of all four BAs were higher in industrial sausages than in artisanal sausages. Tyramine was the major BA detected in 46 of 62 sausages, at a maximum concentration of 785.22 mg/kg. Enterococci and coagulase-negative staphylococci (mainly the meat starter culture Staphylococcus xylosus) were the main tyramine producers. Putrescine was found in 20 of 62 samples, at a maximum concentration of 707.77 mg/kg. Concentrations of these two BAs were significantly correlated (P = 0.0407). Cadaverine and histamine were detected in nine and eight samples, respectively, and both were found in significantly higher concentrations (P = 0.019 and 0.036, respectively) in industrial sausages. Based on the tyramine concentration, five groups of fermented sausages were identified: group 1, very high concentrations (>700 mg/kg); group 2, high concentrations (400 to 700 mg/kg); group 3, moderate concentrations (200 to 400 mg/kg); group 4, low concentrations (<200 mg/kg); group 5, concentrations below the detection limit (0.05 mg/kg). Product samples with tyramine concentrations >200 mg/kg were considered of lower quality because consumption of such samples could be unhealthy for sensitive consumers.

Effect of Blanching Plus Fermentation on Selected Functional Properties of Mealworm (Tenebrio molitor) Powders.

The aim of this study was to determine the effect of blanching followed by fermentation of mealworms (Tenebrio molitor) with commercial meat starter cultures on the functional properties of powders produced from the larvae. Full fat and defatted powder samples were prepared from non-fermented and fermented mealworm pastes. Then the crude protein, crude fat, and dry matter contents, pH, bulk density, colour, water and oil binding capacity, foaming capacity and stability, emulsion capacity and stability, protein solubility, quantity of free amino groups, and protein composition of the powders were evaluated. Regardless of the starter culture used, the blanching plus fermentation process reduced the crude and soluble protein contents of the full fat powders and in general impaired their water and oil binding, foaming, and emulsifying properties. Defatting of the powders improved most functional properties studied. The o-phthaldialdehyde assay revealed that the amount of free amino groups was higher in the fermented powders while sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the soluble proteins of the fermented powders were composed of molecules of lower molecular mass compared to non-fermented powders. As molecular sizes of the soluble proteins decreased, it was clear that the protein structure was also modified by the fermentation process, which in turn led to changes in functional properties. In general, it was concluded that fermentation of mealworms with blanching as a pre-treatment does not contribute to the functional properties studied in this work. Nevertheless, the results confirmed that the properties of non-fermented powders are comparable to other food protein sources.

Meat starter cultures: Isolation and characterization of lactic acid bacteria from traditional sausages

Abstract Fermented meat products represent an important segment of our alimentation. Obtaining these products is based on beneficial microorganism activity. In the case of traditional food products, these are commercial starters or autochthonous microflora. Fermentation of raw materials is mainly done by sugar metabolization of lactic acid bacteria (LAB). In addition, these microorganisms can have other beneficial properties too such as probiotic properties, antimicrobial compound production abilities, etc. In order to meet consumer demands, starter cultures are continuously developed to produce high-quality, healthy, and tasty products, thus contributing to guaranteeing microbiological safety and to improving one or more sensory characteristics, technological, nutritional, or health properties of the fermented products. The aim of our research is to determine the technological properties of autochthonous lactic acid bacteria originated from commercial fresh sausages in order to select and use them as potential starter cultures in the meat industry. In our work, we determined the relevant characteristics (such as salt tolerance, proteolytic activity, antimicrobial activity, and antibiotic resistance) of bacteria isolated from 16 fresh sausages. Based on our results, the studied bacterial isolates originated from sausages could be potentially used as autochthonous meat starter cultures.

Evolution of indigenous starter microorganisms and physicochemical parameters in spontaneously fermented beef, horse, wild boar and pork salamis produced under controlled conditions

The present work was carried out to evaluate the microbiological and physicochemical composition of salamis produced with the meat of beef, horse, wild boar and pork. Salami productions occurred under controlled laboratory conditions to exclude butchery environmental contaminations, without the addition of nitrate and nitrite. All trials were monitored during the ripening (13 °C and 90% relative humidity) extended until 45 d. The evolution of physicochemical parameters showed that beef and pork salamis were characterized by a higher content of branched chain fatty acids (FA) and rumenic acid than horse and wild boar salamis, whereas the last two productions showed higher values of secondary lipid oxidation. Plate counts showed that lactic acid bacteria (LAB), yeasts and coagulase-negative staphylococci (CNS) populations dominated the microbial community of all productions with Lactobacillus and Staphylococcus as most frequently isolated bacteria. The microbial diversity evaluated by MiSeq Illumina showed the presence of members of Gammaproteobacteria phylum, Moraxellaceae family, Acinetobacter, Pseudomonas, Carnobacterium and Enterococcus in all salamis.This study showed the natural evolution of indigenous fermented meat starter cultures and confirmed a higher suitability of horse and beef meat for nitrate/nitrite free salami production due to their hygienic quality at 30 d.

The effect of cranberry pomace ethanol extract on the growth of meat starter cultures, Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis and Listeria monocytogenes

The effect of cranberry pomace extract (CE), on the growth of commonly used meat fermentation starter cultures, and selected foodborne pathogens were studied. Ten meat starter culture strains, belonging to genus Lactobacillus, Pediococcus, and Staphylococcus, and three pathogens, Escherichiacoli O157:H7, Salmonella enterica serovar Enteritidis, and Listeria monocytogenes were grown in either dextrose-free De Man, Rogosa and Sharpe broth, or dextrose-free tryptic soy broth, respectively. Six CE concentrations (0.125, 0.25, 0.50, 0.75, 1.00, and 1.25% wt/vol) were used. Changes in bacterial growth at 37 °C was monitored by measuring absorbance at 600 nm. Concentration-dependent growth stimulation was observed for all starter cultures studied. Lactobacillus spp. and Pediococcus spp. demonstrated maximum stimulatory concentration (MSC) at 0.5–1.00% wt/vol CE. Unlike Lactobacillus spp. and Pediococcus spp., Staphylococcus spp. was more sensitive to CE with complete growth inhibition at concentration of 0.50% wt/vol or higher CE. Reduced growth at higher CE levels could be attributed to the combined effect of inherited acidity and increased phenolic content. All pathogens studied showed a higher sensitivity towards CE than the starter cultures. Findings suggested that CE can potentially be used as a natural antimicrobial against foodborne pathogens and growth promoter for certain meat starter cultures.

Technological and safety characterization of coagulase-negative staphylococci with high protease activity isolated from Traditional Chinese fermented sausages

The aim of this study was to determine the technological and safety properties of coagulase-negative staphylococci (CNS) with high protease activity isolated from traditional Chinese fermented sausages in 10 different regions. According to the criteria of meat starter cultures, eight strains were obtained from 76 Gram-positive and catalase-positive isolates of CNS. These strains could hydrolyze both sarcoplasmic and myofibrillar proteins, tolerate 10% of salts and 150 mg/kg nitrites, possessed good nitrate reductase activity and lipolytic activity, and exhibited non-decarboxylase activity. Furthermore, all the eight strains exhibited antibiotic susceptibilities and negative hemolytic activity, DNase activity, and biofilm formation ability. Whole genome sequencing demonstrated that there were no pathogenic genes in the eight CNS strains, including 18 enterotoxin genes, panton-valentine leukocidin, toxic shock syndrome toxin-1, exfoliative toxin genes, and biofilm genes. Based on the results of API and 16S rRNA sequence analysis, seven of the eight CNS strains were identified as Staphylococcus simulans and the remaining one was S. saprophyticus.

Comparative genomics of Lactobacillus sakei supports the development of starter strain combinations

Strains of Lactobacillus sakei can be isolated from a variety of sources including meat, fermented sausages, sake, sourdough, sauerkraut or kimchi. Selected strains are widely used as starter cultures for sausage fermentation. Recently we have demonstrated that control about the lactic microbiota in fermenting sausages is achieved rather by pairs or strain sets than by single strains. In this work we characterized the pan genome of L. sakei to enable exploitation of the genomic diversity of L. sakei for the establishment of assertive starter strain sets. We have established the full genome sequences of nine L. sakei strains from different sources of isolation and included in the analysis the genome of L. sakei 23K. Comparative genomics revealed an accessory genome comprising about 50% of the pan genome and different lineages of strains with no relation to their source of isolation. Group and strain specific differences could be found, which namely referred to agmatine and citrate metabolism. The presence of genes encoding metabolic pathways for fructose, sucrose and trehalose as well as gluconate in all strains suggests a general adaptation to plant/sugary environments and a life in communities with other genera. Analysis of the plasmidome did not reveal any specific mechanisms of adaptation to a habitat. The predicted differences of metabolic settings enable prediction of partner strains, which can occupy the meat environment to a large extent and establish competitive exclusion of autochthonous microbiota. This may assist the development of a new generation of meat starter cultures containing L. sakei strains.

Screening of lactic acid bacteria with high protease activity from fermented sausages and antioxidant activity assessment of its fermented sausages

ABSTRACTThis study was conducted to screen the lactic acid bacteria (LAB) with admirable technological properties and high protease activity from traditional Chinese fermented sausages, and to investigate the effects of LAB on the antioxidant activity of peptides of fermented sausages. Seven strains were obtained from 133 LAB isolates. They complied with the criteria of meat starter cultures with high proteolytic activity. These strains were analyzed by API and sequence analysis of 16S rRNA to identify the five strains of Lactobacillus plantarum, one strain of L. fermentum, and one strain of L. pentosus. Furthermore, L. plantarum CD101 was inoculated with fermented sausages. LC-MS/MS was used to analyze the peptide composition. Compared with control group, using L. plantarum CD101 as starter cultures (Group L) led to a significant increase (P < 0.05) in antioxidant activity. The relative content of most peptides in Group L samples was largely higher than that in control samples.

Pervasiveness of Staphylococcus carnosus over Staphylococcus xylosus is affected by the level of acidification within a conventional meat starter culture set-up

Staphylococcus carnosus and Staphylococcus xylosus are commonly used, individually or in combination, within conventional starter cultures for the purposes of colour and flavour development during meat fermentation. Yet, little is known about the relative importance of both species under different processing conditions. The present study aimed at investigating the competitiveness of S. carnosus within a meat starter culture under different acidification profiles. The experimental set-up involved a gradient of decreasing experimental control but increasing realism, ranging from liquid meat fermentation models in a meat simulation medium, over solid mince-based meat fermentation models, to fermented sausage production on pilot-scale level. In general, S. carnosus gained a fitness advantage over S. xylosus in the most acidified variants of each set-up. In contrast, increasing persistence of S. xylosus was seen at the mildest acidification profiles, especially when approximating actual meat fermentation practices. Under such conditions, S. carnosus was reduced to co-prevalence in the mince-based meat fermentation models and was fully outcompeted on pilot-scale level. The latter was even the case when no S. xylosus starter culture was added, whereby S. carnosus was overpowered by staphylococci that originated from the meat background (mostly S. xylosus strains). The results of the present study suggested that conventional starter cultures behave differently when applied in different technological set-ups or using different recipes, with possible repercussions on fermented meat product quality.

Lactic acid bacteria isolated from raw and fermented pork products: Identification and characterization of catalase-producing Pediococcus pentosaceus.

Lactic acid bacteria (LAB) from raw and fermented pork samples were screened for their inhibitory activity by an agar spot test in order to obtain a LAB strain with suitable property to be used as meat starter cultures. Among the 174 isolates, 73 were positive to inhibit at least one of the seven indicator bacteria, which were further characterized. The most suitable isolate was isolate P0805, identified as Pediococcus pentosaceus. This bacterium was catalase- and nitrate reductase-positive and amino acid decarboxylase-negative; moreover, it produced inhibitory substances against Salmonella Typhimurium with the activity of the partially purified inhibitory substances of 409,600 AU/mL. To further characterize the catalase-producing ability of P. pentosaceus P0805, the effect of hematin on its catalase activity in Sausage Model Broth (SMB) was evaluated, and it enhanced catalase production. The catalase activity was found in both SMB with and without hematin. It was concluded that catalase produced by this bacterium was heme-independent catalase.

Genetic diversity and population structure of food-borne Staphylococcus carnosus strains

The species Staphylococcus carnosus is a non-pathogenic representative of the coagulase negative staphylococci. Specific strains are applied as meat starter cultures. The species consists of two subspecies, S. carnosus ssp. carnosus and S. carnosus ssp. utilis. In order to place S. carnosus strains, characterized in former studies, in a genetic background that allows a typing of candidates for starter cultures, a Multilocus Sequence Typing (MLST) scheme was developed. Internal fragments of the genes tpiA, xprT, dat, gmk, glpK, narG, cstA, encoding triosephosphate isomerase, xanthine phosphoribosyltransferase, d-amino acid aminotransferase, guanylate kinase, glycerol kinase, the α-chain of the respiratory nitrate reductase, and a carbon starvation protein where chosen. Genes were selected based on their equal distribution in the genome, taxonomic value in subspecies differentiation and metabolic function. This MLST was applied to 44 S. carnosus strains, most of them previously analyzed for their suitability as starter cultures.The number of alleles varied between zero (tpiA) and five (cstA) and allowed the definition of nine sequence types (ST). ST1 was most abundant (18 strains), followed by ST2 (8) and ST4 (6). The nine STs confirmed a close relationship of all strains despite their isolation source and year, but lacked correlation with physiological activities relevant for starter cultures. The low amount of STs in the strain set lets us suggest that recombination between strains is rare. Thus, it is hypothesized that evolutionary events seem to be due to single point mutations rather than intrachromosomal recombination, and that the species possesses a clonal population structure.

Complete Genome Sequence of Staphylococcus carnosus LTH 3730.

Specific strains of the apathogenic coagulase-negative species Staphylococcus carnosus are frequently used as meat starter cultures, as they contribute to color formation and the production of aroma compounds. Here, we report the complete genome sequence of S. carnosus LTH 3730, a strain isolated from a fermented fish product.

The Peptidoglycan Pattern of Staphylococcus carnosus TM300-Detailed Analysis and Variations Due to Genetic and Metabolic Influences.

The Gram-positive bacterium Staphylococcus carnosus (S. carnosus) TM300 is an apathogenic staphylococcal species commonly used in meat starter cultures. As with all Gram-positive bacteria, its cytoplasmic membrane is surrounded by a thick peptidoglycan (PGN) or murein sacculus consisting of several layers of glycan strands cross-linked by peptides. In contrast to pathogenic staphylococci, mainly Staphylococcus aureus (S. aureus), the chemical composition of S. carnosus PGN is not well studied so far. UPLC/MS analysis of enzymatically digested S. carnosus TM300 PGN revealed substantial differences in its composition compared to the known pattern of S. aureus. While in S. aureus the uncross-linked stem peptide consists of a pentapeptide, in S. carnosus, this part of the PGN is shortened to tripeptides. Furthermore, we found the PGN composition to vary when cells were incubated under certain conditions. The collective overproduction of HlyD, FtsE and FtsX—a putative protein complex interacting with penicillin-binding protein 2 (PBP2)—caused the reappearance of classical penta stem peptides. In addition, under high sugar conditions, tetra stem peptides occur due to overflow metabolism. This indicates that S. carnosus TM300 cells adapt to various conditions by modification of their PGN.

Degradation of PCBs in dry fermented sausages during drying/ripening

The effects of several commercial meat starter cultures on degradation of polychlorinated biphenyls (PCBs) in dry fermented sausages over 28days of drying/ripening were investigated. The sausage batter was prepared according to a classic recipe and spiked with a standard solution of a PCB congener mixture. With addition of different commercial meat starter cultures, five experimental groups were prepared: no further addition; and separate addition of each of four starter cultures: Texel DCM-1, Texel LM-30, Biostar Sprint, and SM-181. Samples were taken at the beginning of fermentation (zero time), and after 4, 7, 14, 21 and 28days. PCB residues were extracted with hexane. The PCB contents were determined using gas chromatography–mass spectrometry. The PCB levels were reduced in all of the experimental groups tested, where addition of starter culture Biostar Sprint (Lactobacillus sakei, Staphylococcus carnosus, Staphylococcus xylosus) showed the highest PCB degradation rates.