Terminal-restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis profiling of fungal communities in Camembert cheese

Identifying and isolating yeasts and moulds within a fungal community is challenging. The main goal of the present study was to assess a new approach for the detection and identification of fungi involved in Camembert cheese rind formation to replace the use of traditional microbiological techniques. Two molecular methods, terminal-restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA), were adapted and compared for their potential to determine fungal composition directly from cheese samples. The two techniques in combination with principal component analysis showed differences in the fungal composition of cheeses when comparing surface with centre, different batches, manufacturing processes and ripening times. Moreover, cheese stabilisation induced changes in the flora at the cheese centre, and difference in size (150 g versus 1 kg) modified surface flora. Nine fungal genera were identified in cheese samples: Cladosporium, Debaryomyces, Geotrichum, Kluyveromyces, Mucor, Penicillium, Pichia, Saccharomyces and Yarrowia.     

Lactobacillus amylovorus DSM 19280 as a novel food-grade antifungal agent for bakery products

Mould spoilage is the main cause of substantial economic loss in bakery industry and might also cause public health problems due to the production of mycotoxins. The reduction of mould growth in bakery products is thus of crucial importance and there is great interest to develop safe and efficient strategies for this purpose. In this study Lactobacillus amylovorus DSM19280 has been shown to produce a wide spectrum of antifungal compounds active against common bread spoilage fungi. Among the indicator moulds, Aspergillus fumigatus and Fusarium culmorum were the most sensitive organisms. Several antifungal compounds were found to be present in synthetic medium inoculated with L. amylovorus DSM19280 strain, some of them being reported here for the first time. Wheat doughs fermented with L. amylovorus DSM19280 had good rheological properties and the breads thereof were of high quality as shown by rheofermentometer and texture analyser measurements. The results were compared with those obtained with a control non-antifungal L. amylovorus DSM20531^T strain, a non-acidified and a chemically acidified dough. The quality of sourdough and bread fermented with L. amylovorus DSM 19280 was comparable to that obtained by using L. amylovorus DSM20531 ^T. Additionally, breads were evaluated for the ability to retard the growth of Fusarium culmorum FST 4.05, Aspergillus niger FST4.21, Penicillium expansum FST 4.22, Penicillium roqueforti FST 4.11 and fungal flora from the bakery environment. The biological preservation of bread with L. amylovorus DSM 19280 was also compared to the most commonly used antifungal agent Calcium propionate. Breads containing sourdough fermented with L. amylovorus DSM 19280 were more effective in extending the shelf life of bread than the calcium propionate.     

Filamentous Fungi and Mycotoxins in Cheese: A Review

Important fungi growing on cheese include Penicillium, Aspergillus, Cladosporium, Geotrichum, Mucor, and Trichoderma. For some cheeses, such as Camembert, Roquefort, molds are intentionally added. However, some contaminating or technological fungal species have the potential to produce undesirable metabolites such as mycotoxins. The most hazardous mycotoxins found in cheese, ochratoxin A and aflatoxin M1, are produced by unwanted fungal species either via direct cheese contamination or indirect milk contamination (animal feed contamination), respectively. To date, no human food poisoning cases have been associated with contaminated cheese consumption. However, although some studies state that cheese is an unfavorable matrix for mycotoxin production; these metabolites are actually detected in cheeses at various concentrations. In this context, questions can be raised concerning mycotoxin production in cheese, the biotic and abiotic factors influencing their production, mycotoxin relative toxicity as well as the methods used for detection and quantification. This review emphasizes future challenges that need to be addressed by the scientific community, fungal culture manufacturers, and artisanal and industrial cheese producers.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Formation of alanine, α-aminobutyrate,acetate, and 2-butanol during cheese ripening by Pediococcus acidilactici FAM18098

There is limited information about the contribution of Pediococcus acidilactici, a nonstarter lactic acid bacteria, to cheese ripening and flavour development. Model Tilsit-type and Gruyère-type cheeses were produced using P. acidilactici FAM18098 as an adjunct. The adjunct did not influence the cheese manufacturing processes. The pediococcal log counts ranged from 7.0 to 8.0 cfu g⁻¹ after 90 and 120 days of ripening. P. acidilactici produced ornithine, a result of arginine metabolism by the arginine deiminase pathway, and α-aminobutyrate and alanine while simultaneously metabolising serine and threonine. The analysis of the volatile compounds in the cheeses showed that higher acetate, 2-butanone, and 2-butanol levels and lower diacetyl levels were present in the cheeses produced with P. acidilactici than in the control cheeses. The study illustrates that P. acidilactici can influence amino acid metabolism in cheese; further, ornithine, α-aminobutyrate, and acetate can serve as indicators for the presence of this species.     

Biogenic amine content and proteolysis in Manchego cheese manufactured with Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

Two different autochthonous strain starter cultures, in which the acidifying starter was composed of strains of Lactococcus lactis, were used for the manufacture of pasteurised milk Manchego cheese. Proteolysis parameters, biogenic amines and sensory characteristics were evaluated and compared with those of commercial starter Manchego cheese and raw milk Manchego cheese manufactured without starter. Autochthonous starter cheeses, and especially those including Lactobacillus paracasei subsp. paracasei as adjunct, presented higher levels of proteolysis than in commercial starter cheese. The concentrations of total biogenic amines in autochthonous starter cheeses were much lower than in raw milk cheese and even lower than in commercial starter cheese. Cheese manufactured with the adjunct strain gave the best results for both flavour intensity and flavour quality, and was the most preferred by panellists. The results suggest that the culture containing Lb. paracasei subsp. paracasei as adjunct could be used for the manufacture of industrial Manchego cheese.     

Volatile composition and improvement of the aroma of industrial Manchego cheese by using Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

The use of several autochthonous strains of lactic acid bacteria, including Lactobacillus paracasei subsp. paracasei as adjunct of the starter in the manufacture of Manchego cheese, was evaluated in an attempt to improve the aroma of the industrial Manchego cheese. Volatile composition and odour characteristics were evaluated and compared to those in Manchego cheese manufactured with a commercial starter (CS) culture and with raw milk cheese manufactured without starter. Manchego cheeses manufactured with two autochthonous strains of Lactococcus lactis subsp. lactis displayed a similar volatile profile and odour characteristics to the cheese made with the CS. The use of the strain Lactobacillus paracasei subsp. paracasei CECT 7882 as adjunct of the Lactococcus strains produced cheeses with higher amounts of some free fatty acids and alcohols, acetoin, lactones, phenylacetaldehyde, 2-phenylethanol and linalool, and higher scores of the odour intensity, odour quality, and ewe’s milk odour than the CS cheeses. It resulted in an intensification and improvement of industrial Manchego cheese aroma.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.     

Influence of bacteria used as adjunct culture and sunflower oil addition on conjugated linoleic acid content in buffalo cheese

The influence of bacteria and sunflower oil addition on conjugated linoleic acid content (CLA) in buffalo cheese was determined. Fresh and short-ripened cheeses were manufactured using the same starter culture and four different adjunct strains previously selected by their CLA production rate. Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum and Streptococcus thermophilus were individually used as adjunct culture. Sunflower oil (SO) was added to obtain a final concentration of 200 μg/ml of linoleic acid. CLA levels in cheese were higher than raw milk, especially after ripening time. SO supplementation increase CLA concentrations in fresh cheeses, except in those manufactured with S. thermophilus as adjunct culture. Both, ripening and SO supplementation showed a positive influence on CLA concentration. Similar texture, acidity and colour were determined in cheeses with or without SO supplementation. Buffalo cheeses manufactured with appropriate adjunct cultures may be a natural source of CLA for human consumption.     

Dynamics of starter,adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.     

Inhibitory effect of cinnamon essential oils on selected cheese-contaminating fungi (Penicillium spp.) during the cheese-ripening process

The antifungal effects of essential oils (EOs) on cultures of the known cheese fungal contaminants Penicillium spp. were evaluated. Cinnamon leaf and bark EOs were the most effective among 8 EOs tested. The main components of both cinnamon EOs were eugenol, cinnamaldehyde, and linalool. Both inhibited growth of Penicillium spp. at a concentration of 10% v/v (2,000 ppm/mm²) on a lawn cell plate. When tested using 3 commercially available cheese starters, cinnamon EOs showed no effect against the FD-DVS ABT-5 cheese starter. However, growth of lactobacilli was inhibited in the presence of ≥10% (v/v) of leaf and bark EOs for the KAZU 1 starter, and ≥5% (v/v) for the FD-DVS FLORA-DANICA starter. A concentration of 4,000 ppm/mm² of cinnamon EOs completely inhibited growth of the Penicillium spp. that naturally contaminates the surface of Appenzeller cheese.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

Effect of high-pressure-processing on the microbiology,proteolysis, texture and flavour of Brie cheese during ripening and refrigerated storage

Brie cheeses were high pressure (HP)-treated at 400 or 600 MPa on days 14 or 21 after manufacture to prevent over-ripening. Lactic acid bacteria and Penicillium camemberti numbers declined markedly after HP treatment. In control cheese pH increased 2.0 units from day 21 to day 60, but less than 0.3 units in HP-treated cheeses. Cheeses treated at 600 MPa showed the maximum concentrations of residual caseins during refrigerated storage and control cheese the minimum concentrations. A 7.6-fold increase in hydrophobic peptides was recorded from day 21 to day 60 in control cheese and 0.8–1.6-fold increases in HP-treated cheeses. The maximum aminopeptidase activity was detected in control cheese, the highest free amino acid concentrations in cheeses treated at 400 MPa. The firmest texture was recorded for cheeses treated on day 14 at 400 or 600 MPa. HP-treated cheeses showed higher flavour quality scores than control cheese from day 60 onwards.     

Influence of microfiltration and adjunct culture on quality of Domiati cheese

The effects of microfiltration and pasteurization processes on proteolysis, lipolysis, and flavor development in Domiati cheese during 2 mo of pickling were studied. Cultures of starter lactic acid bacteria isolated from Egyptian dairy products were evaluated in experimental Domiati cheese for flavor development capabilities. In the first trial, raw skim milk was microfiltered and then the protein:fat ratio was standardized using pasteurized cream. Pasteurized milk with same protein:fat ratio was also used in the second trial. The chemical composition of cheeses seemed to be affected by milk treatment—microfiltration or pasteurization—rather than by the culture types. The moisture content was higher and the pH was lower in pasteurized milk cheeses than in microfiltered milk cheeses at d 1 of manufacture. Chemical composition of experimental cheeses was within the legal limits for Domiati cheese in Egypt. Proteolysis and lipolysis during cheese pickling were lower in microfiltered milk cheeses compared with pasteurized milk cheeses. Highly significant variations in free amino acids, free fatty acids, and sensory evaluation were found among the cultures used in Domiati cheesemaking. The cheese made using adjunct culture containing Lactobacillus delbrueckii ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus casei, Lactobacillus plantarum, and Enterococcus faecium received high scores in flavor acceptability. Cheeses made from microfiltered milk received a higher score in body and texture compared with cheeses made from pasteurized milk.     

The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production

Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of all microbial groups examined initially increased, but declined after Baski scalding and remained practically stable up to day 90. The presence of S. macedonicus was confirmed until the end of ripening (Cheeses B and C); this was also the case for the bacteriocin produced by S. macedonicus ACA-DC 198. X-propyl-dipeptidyl-aminopeptidase activity was detected in all cheeses after day 15. The physicochemical characteristics of the mature Cheese C were in agreement with those defined by the Greek legislation. The sensory properties of all mature cheeses corresponded with the ones characterizing the traditional Kasseri cheese. It was concluded that S. macedonicus ACA-DC 198 can be used as an adjunct in Kasseri cheese production.     

The Effect of Yeast Species from Raw Milk in China on Proteolysis and Aroma Compound Formation in Camembert-Type Cheese

The influence of selected yeast strains (Pichia fermentans, Issatchenkia orientalis, Yarrowia lipolytica) on Camembert-type cheeses has been investigated by monitoring their microbial and compositional changes. During the ripening period from day 1 to day 15, the yeast strains grew exponentially and then slowed to a moderate growth rate. Our results indicated that the selected strains did not significantly affect the general characteristics of cheese but rather the content and ratio of individual free amino acids (P < 0.05). In the cheese with added I. orientalis, a greater amount of small peptides and a higher concentration of non-protein nitrogen and NH₃ were found. Meanwhile, the presence of adjunct yeasts did not enhance the lipolysis activity in cheese. A principal component analysis of 30 free fatty acids and volatile compounds differentiated five cheeses out of eight according to the content of specific free fatty acids and volatile compounds. In terms of the ripening period, two groups of cheeses at 15 and 45 days were distinguished.     

Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese

The effect of two commercially available adjunct cultures, LBC 80 (Lactobacillus casei subsp. rhamnosus) and CR-213 (containing Lactococcus lactis subsp. cremoris and Lc. lactis subsp. lactis) on the proteolysis in low-fat hard ewes’ milk cheese of Kefalograviera-type was investigated. Two controls, a full-fat cheese (306 g kg⁻¹ fat, 378 g kg⁻¹ moisture) and a low-fat cheese (97 g kg⁻¹ fat, 486 g kg⁻¹ moisture, made using a modified procedure), were also prepared. The effect of adjunct culture on proteolysis, as examined by polyacrylamide gel electrophoresis of cheese and water soluble cheese extracts, was marginal. The reverse-phase HPLC peptide profiles of the water soluble extracts from low-fat cheeses were similar although some quantitative differences were observed between low-fat control cheese and experimental cheeses. The fat content as reflected by the differences in peptide profiles affected the pattern of proteolysis. Proteolysis, as measured by the percentage of total nitrogen soluble in water or in 120 g L⁻¹ trichloroacetic acid, was significantly (P<0.05) affected by the addition of adjunct cultures. Furthermore, the adjunct cultures enhanced the production of low molecular mass nitrogenous compounds; the levels of total nitrogen, soluble in 50 g L⁻¹ phosphotungstic acid, and of free amino acids were significantly (P<0.05) higher in the low-fat experimental cheeses than in the low-fat control cheese.