Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Inhibitory Effect of PgAFP and Protective Cultures on Aspergillus parasiticus Growth and Aflatoxins Production on Dry-Fermented Sausage and Cheese.

Aflatoxigenic molds can grow and produce aflatoxins on dry-fermented meat and cheese. The small, basic, cysteine-rich antifungal protein PgAFP displays a time-limited inhibitory ability against unwanted molds by increasing reactive oxygen species (ROS), which can lead to increased aflatoxin production. However, calcium abolishes the inhibitory effect of PgAFP on certain Aspergillus spp. To maximize the antifungal effect, this protein may be combined with protective cultures. Yeasts and lactic acid bacteria may counteract the impact of calcium on PgAFP fungal inhibition. The objective of this work was to study the effect of PgAFP and different combined treatments with Debaryomyces hansenii and/or Pediococcus acidilactici against growth of and aflatoxin production by an aflatoxigenic strain of Aspergillus parasiticus in both culture media and dry-fermented foods with low or high calcium levels. Aflatoxins production was increased by PgAFP but dramatically reduced by P. acidilactici in low calcium culture medium, whereas in the Ca-enriched culture medium, all treatments tested led to low aflatoxins levels. To study whether PgAFP and the protective microorganisms interfere with ROS and aflatoxin production, the relative expression of genes foxA, which is involved in peroxisomal ß-oxidation, and aflP, which is required for aflatoxin biosynthesis, were evaluated. The aflatoxin overproduction induced by PgAFP seems not to be linked to peroxisomal ß-oxidation. The combination of PgAFP and D. hansenii provided a successful inhibitory effect on A. parasiticus growth as well as on aflatoxin production on sliced dry-fermented sausage and cheese ripened up to 15 days, whereas P. acidilactici did not further enhance the protective effect of the two former agents. Therefore, the combined treatment of PgAFP and D. hansenii seems to provide a promising protective mean against aflatoxin-producing A. parasiticus on dry-fermented foods.

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products.

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods.

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include 'omics' approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex ß subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products.

Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP.

Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a ß subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit ß CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

Growth inhibition and stability of PgAFP from Penicillium chrysogenum against fungi common on dry-ripened meat products.

Dry-ripened foods favor the development of a superficial fungal population that may include toxigenic molds. To combat unwanted molds, an antifungal protein from Penicillium chrysogenum (PgAFP) can be useful. The aim of the present work was to study the antimicrobial activity of PgAFP against microorganisms common in dry-ripened foods, and to evaluate its sensitivity to proteolytic enzymes and heat treatments that may be applied to foods, as well as to different pH values. The inhibitory effect of the purified protein on 38 microbial strains grown in culture medium was determined. PgAFP sensitivity to various proteases, heat treatments, and preincubation at different pH values was tested by means of the residual activity on selected reference strains. Inhibitory activity of PgAFP against unwanted molds was tested in a dry-fermented sausage. This protein exhibited potent inhibitory activity against unwanted molds, including the main mycotoxin-producing species of Aspergillus and Penicillium of concern for dry-ripened foods. PgAFP withstood most proteases, intense heat and a wide range of pH values. PgAFP efficiently reduced counts of A. flavus and P. restrictum inoculated on a dry-fermented sausage. This protein can be of interest to control hazardous molds in dry-ripened foods.

Development of real-time PCR methods to quantify patulin-producing molds in food products.

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.

Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum.

The protein PgChP is a new chitosanase produced by Penicillium chrysogenum AS51D that showed antifungal activity against toxigenic molds. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation, only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two isoforms. Analysis of the molecular mass by electrospray ionization-mass spectrometry revealed six major peaks from 30 to 31 kDa that are related to different levels of glycosylation. The pgchp gene has 1,146 bp including four introns and an open reading frame encoding a protein of 304 amino acids. The translated open reading frame has a predicted mass of 32 kDa, with the first 21 amino acids comprising a signal peptide. Two N glycosylation consensus sequences are present in the protein sequence. The deduced sequence showed high identity with fungal chitosanases. A high level of catalytic activity on chitosan was observed. PgChP is the first chitosanase described from P. chrysogenum. Given that enzymes produced by this mold species are granted generally recognized as safe status, PgChP could be used as a food preservative against toxigenic molds and to obtain chitosan oligomers for food additives and nutraceuticals.

Characterization of the novel antifungal protein PgAFP and the encoding gene of Penicillium chrysogenum.

The strain RP42C from Penicillium chrysogenum produces a small protein PgAFP that inhibits the growth of some toxigenic molds. The molecular mass of the protein determined by electrospray ionization mass spectrometry (ESI-MS) was 6 494Da. PgAFP showed a cationic character with an estimated pI value of 9.22. Upon chemical and enzymatic treatments of PgAFP, no evidence for N- or O-glycosylations was obtained. Five partial sequences of PgAFP were obtained by Edman degradation and by ESI-MS/MS after trypsin and chymotrypsin digestions. Using degenerate primers from these peptide sequences, a segment of 70bp was amplified by PCR from pgafp gene. 5'- and 3'-ends of pgafp were obtained by RACE-PCR with gene-specific primers designed from the 70bp segment. The complete pgafp sequence of 404bp was obtained using primers designed from 5'- and 3'-ends. Comparison of genomic and cDNA sequences revealed a 279bp coding region interrupted by two introns of 63 and 62bp. The precursor of the antifungal protein consists of 92 amino acids and appears to be processed to the mature 58 amino acids PgAFP. The deduced amino acid sequence of the mature protein shares 79% identity to the antifungal protein Anafp from Aspergillus niger. PgAFP is a new protein that belongs to the group of small, cysteine-rich, and basic proteins with antifungal activity produced by ascomycetes. Given that P. chrysogenum is regarded as safe mold commonly found in foods, PgAFP may be useful to prevent growth of toxigenic molds in food and agricultural products.

Selection of antifungal protein-producing molds from dry-cured meat products.

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillusniger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicilliumchrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37kDa for AS51D and 9kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.

Production of secondary metabolites by some terverticillate penicillia on carbohydrate-rich and meat substrates.

Most terverticillate penicillia isolated from dry-cured meat products are toxigenic, but their ability to produce hazardous metabolites on meat-based substrates is not well known. The production of extrolites by selected terverticillate penicillia isolated from dry-cured ham has been studied on carbohydrate-rich media (malt extract agar, Czapek yeast autolysate agar, rice extract agar, and rice), meat extract triolein salt agar, and ham slices. Chloroform extracts from the selected strains grown on malt extract agar were toxic for the brine shrimp (Artemia salina) larvae and VERO cells at a concentration of 2 mg/ml, but 0.02 mg/ml produced no toxic effect. Analysis by high-pressure liquid chromatography (HPLC) coupled with photodiode array detection (DAD) or with mass spectrometry (MS) and an atmospheric pressure chemical ionization (APCI) source revealed different biologically active metabolites: cyclopiazonic acid and rugulovasine A from Penicillium commune; verrucosidin, anacine, puberuline, verrucofortine, and viridicatols from Penicillium polonicum; arisugacin and viridicatols from Penicillium echinulatum; and compactin and viridicatols from Penicillium solitum. Most of these metabolites, including the amino acid-derived compounds, were produced in the media containing high levels of carbohydrates. High concentrations of nitrogen compounds in the medium does not imply a greater production of the metabolites studied, not even those derived from the amino acids. However, molds growing on dry-cured ham are able to synthesize limited amounts of some secondary metabolites, a fact not previously reported. The combination of HPLC coupled with DAD and MS-APCI was useful for identification of closely related terverticillate Penicillium species from dry-cured ham. These techniques could be used to characterize the risk associated with the potential production of secondary metabolites in cured meats.

Contribution of a selected fungal population to the volatile compounds on dry-cured ham.

Dry-cured ham is obtained after several months of ripening. Different fungi strive on the surface, including toxigenic molds. Proteolysis and lipolysis by the endogenous and microbial enzymes seem to play a decisive role in the generation of flavor precursors in dry-cured meat products. In addition, fungi show a positive impact on the volatile compounds of ripened pork loins. However, the contribution of the fungal population to flavor formation in dry-cured ham remains unclear. One selected strain each of Penicillium chrysogenum and Debaryomyces hansenii was inoculated as starter cultures on dry-cured ham. Volatile compounds extracted by solid phase micro-extraction technique were analyzed by gas chromatography/mass spectrometry. A trained panel evaluated flavor and texture of fully ripened hams. The wild fungal population on non-inoculated control hams correlates with higher levels of short chain aliphatic carboxylic acids and their esters, branched carbonyls, branched alcohols, and some sulfur compounds, particularly at the outer muscle. Conversely, P. chrysogenum and D. hansenii seem to be responsible for higher levels of long chain aliphatic and branched hydrocarbons, furanones, long chain carboxylic acids and their esters. The very limited impact of P. chrysogenum on pyrazines in inoculated hams can be due to the activity of the yeast. Lower levels for some of the more volatile linear carbonyls at the ham surface suggest an anti-oxidant effect by micro-organisms. The differences in volatile compounds did not show a neat impact on flavor in the sensorial analysis. Nonetheless, inoculated hams got a better overall acceptability, which has to be attributed to their improved texture. The lower toughness of inoculated hams is a direct consequence of an early settling of a highly proteolytic mold. Thus, the use of selected fungi as starter cultures may be useful to obtain high-quality and safe dry-cured ham.

Contribution of a selected fungal population to proteolysis on dry-cured ham.

The proteolytic changes taking place in dry-cured hams lead to increases in free amino acids. Such free amino acids not only contribute to flavour, but also serve as precursors of volatile compounds. Several months of ripening time are required to allow the particular flavour to develop. The fungal population allowed to grow on the surface of some types of dry-cured could play a key role on proteolysis, as it has been shown for dry-cured sausages. The purpose of this work was to study the possible contribution of fungi to proteolysis in dry-cured ham. For this, a strain each of non-toxigenic Penicillium chrysogenum (Pg222) and Debaryomyces hansenii (Dh345), selected for their proteolytic activity on myofibrillar proteins, were inoculated as starter cultures. Changes in the high ionic strength-soluble proteins of an external muscle (adductor) revealed in only 6 months higher proteolysis in the inoculated hams when compared to non-inoculated control hams. Proteolytic strains among the wild fungal population on non-inoculated control hams prevented from obtaining similar differences at the end of processing. However, inoculation with Pg222 and Dh345 led to higher levels for most free amino acids at the external muscle in fully dry-cured hams. In addition, the concentration for some of the more polar free amino acids (i.e. Asp, Glu, Ser and Gln) in inoculated hams was higher at external than at internal (biceps femoris) muscles. These promising results deserve further studies to know the impact of a selected fungal population on the volatile compounds and sensory properties of dry-cured ham.

Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins.

Moulds grow on many different dry-cured meat products and are able to hydrolyse muscle proteins. However, their contribution to proteolysis in these products is not well known. Only recently, the ability of just a few strains of Penicillium spp. to increase proteolysis in dry-cured meat products has been shown. For these strains to be used as starter cultures, their hydrolytic activity under standard conditions should be characterised. With this purpose, the effect of Penicillium chrysogenum Pg222 on pork myofibrillar proteins has been assayed in a culture medium containing 5% (w/v) NaCl. SDS-PAGE revealed that Pg222 was responsible for extensive hydrolysis of the main myofibrillar proteins except alpha-actinin. The proteolysis led to increases in free amino acids, reaching peak values at 84 h. Ala, Tyr and Lys were present in the greatest amount. These results suggest that P. chrysogenum Pg222 would contribute to development of desired texture and flavours in dry-cured meat products.

Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins.

During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development. However, their contribution to volatile components of dry-cured meat products is not known. One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening. Sterile pork loins were inoculated and ripened for 106 days. Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS. Inoculation of pork loins with P. chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids. Inoculation with D. hansenii seemed to favour the formation of complex alcohols.

Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat extract-based substrate.

Penicillium commune, a mold frequently found on dry-cured meat products, is able to synthesize the mycotoxin cyclopiazonic acid (CPA). To evaluate the hazard due to CPA on such foods, the ability of P. commune to grow and produce CPA at water activities (a(w)) in the range of 0.99 to 0.90 with a meat extract-based medium from 12 to 30 degrees C was determined. CPA was quantified by high-pressure liquid chromatography and mass spectrometry. P. commune was able to grow at every a(w) and temperature tested. The optimal environmental conditions for growth were 20 to 25 degrees C, at 0.97 to 0.96 a(w), but the highest amount of CPA was produced at 30 degrees C, 0.96 a(w). No direct correlation between growth rate and CPA production was assessed. Temperature seems to be the most important factor influencing CPA production. However, there was an interaction between temperature and a(w) that significantly (P < 0.001) affected growth and CPA production. An a(w) of 0.90 had a marked effect, depressing growth and CPA production. Meat extract-based medium proved to be an appropriate substrate for CPA biosynthesis by P. commune under a wide range of conditions.

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

Proteolytic activity of Penicillium chrysogenum and Debaryomyces hansenii during controlled ripening of pork loins.

The role of micro-organisms on the ripening process of dry-cured ham, particularly with respect to proteolysis, is not clear. This is partially due to the lack of an adequate system to study changes on a sterile control meat product for long ripening times. Using a meat system based on sterile pork loins ripened under aseptic conditions for 106 days, the contribution to the proteolysis of two micro-organisms isolated from dry-cured ham has been established. Changes were studied by SDS-PAGE of sarcoplasmic and myofibrillar proteins, capillary zone electrophoresis (CZE) of low ionic strength-soluble nitrogen compounds, and HPLC of free amino acids. Debaryomyces hansenii Dh345 did not show any significant proteolytic activity. However, Penicillium chrysogenum Pg222 showed high proteolytic activity on myofibrillar proteins resulting in an increase in soluble nitrogen compounds. For this, P. chrysogenum Pg222 should be considered to be used as starter culture in meat products made using long ripening times.

Effect of Carbon Dioxide and Oxygen Enriched Atmospheres on the Shelf-Life of Refrigerated Pork Packed in Plastic Bags.

Changes in bacterial numbers, metmyoglobin percentage and 2-thiobarbituric acid number during the chill storage of pork longissimus dorsi packed with air, carbon dioxide, carbon dioxide and oxygen or vacuum-packed in plastic bags of high (polyethylene) and low (Cryovac BB-1) permeability to gases were studied. The fast increase of viable counts in polyethylene bags showed that plastic films of high permeability are not suitable to extend the shelf-life of meat using modified atmospheres. In Cryovac bags both carbon dioxide-enriched atmospheres and vacuum-packaging inhibited aerobic organisms, keeping the bacterial flora (mainly lactobacilli and Brochothrix thermosphacta ) below the level of 108/cm2 for about 3 weeks. Metmyoglobin formation was not affected by 20% carbon dioxide, whilst 80% oxygen significantly decreased its rate. Metmyoglobin accumulation in vacuum-packed samples was even slower than in the oxygen-enriched atmosphere, but meat color was less attractive in the former than in the latter. Lipid oxidation was not the limiting factor of shelf-life in either of these two atmospheres. Off-odors appeared in both at approximately 20-22 d of storage.