Impact of Gram-negative bacteria in interaction with a complex microbial consortium on biogenic amine content and sensory characteristics of an uncooked pressed cheese

The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines (BA) in the core of an uncooked pressed type model cheese was investigated in the presence of a defined complex microbial consortium. Eleven strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA-production ability, were individually tested in a model cheese. Four out of 6 strains of Enterobacteriaceae (Citrobacter freundii UCMA 4217, Klebsiella oxytoca 927, Hafnia alvei B16 and Proteus vulgaris UCMA 3780) reached counts close to 6 log CFU g⁻¹ in the model cheese. In core of cheeses inoculated with Gram-negative bacteria, only slight differences were observed for microbial counts (Enterococcus faecalis or Lactobacillus plantarum count differences below 1 log CFU g⁻¹), acetate concentration (differences below 200 mg kg⁻¹) and texture (greater firmness) in comparison to control cheeses. Cheese core colour, odour and volatile compound composition were not modified. Although ornithine, the precursor of putrescine, was present in all cheeses, putrescine was only detected in cheeses inoculated with H. alvei B16 and never exceeded 2.18 mmol kg⁻¹ cheese dry matter. Cadaverine was only detected in cheeses inoculated with H. alvei B16, K. oxytoca 927, Halomonas venusta 4C1A or Morganella morganii 3A2A but at lower concentrations (<1.05 mmol kg⁻¹ cheese dry matter), although lysine was available. Only insignificant amounts of the detrimental BA histamine and tyramine, as well as isopentylamine, tryptamine or phenylethylamine, were produced in the cheese model by any of the Gram-negative strains, including those which produced these BA at high levels in vitro.     

Contribution of coagulant and native microflora to the volatile-free fatty acid profile of an artisanal cheese

The contributions of the coagulant Cynara cardunculus and of the microflora of raw milk to the volatile-free fatty acid profile of Serra da Estrela cheese were evaluated. The experimental design included both a model system and, dual cheeses. The study in the model system showed that isovaleric acid was the predominant volatile compound after 7 d of ripening. The systems inoculated with Enterococcus faecium produced the highest amount of this volatile (ca. 135.8 mg kg⁻¹ curd), while those inoculated with Lactobacillus plantarum produced the least (21.4 mg kg⁻¹ curd); Lactococcus lactis produced moderate amounts (ca. 34.2 mg kg⁻¹ curd) but a total amount of volatile-free fatty acids similar to those found in control samples. This is considered advantageous since this volatile fatty acid confers a harsh, piquant, mature flavour to cheese, coupled with the realisation that excess volatiles may result in off-flavours. The addition of cultures in experimental cheeses helped reduce ripening time to about one half. Inclusion of Lb. plantarum led to cheeses containing the highest amounts of volatiles, and exhibiting an aroma closest to that of typical Serra da Estrela cheese.     

Study of Enterobacteriaceae during the manufacture and ripening of San Simón cheese

The aim of this work was to study the evolution of the population of Enterobacteriaceae in one of the traditional Spanish cheeses, San Simón cheese, during the manufacture and ripening processes and its interrelation with the changes in some physico–chemical parameters.The evolution of the Enterobacteriaceae counts (VRBGA medium) and coliform counts (VRBA medium) was studied from samples of milk, curd and inner and surface zones of the cheese at different stages of ripening from five batches of traditionally manufactured artisan cheese. The counts obtained were very similar in both media and in general one log unit higher in the inner portion of the cheeses than on the surface.TheEnterobacteriaceae counts in milk were 10²–10³cfu g⁻¹and the counts increased during the first week of ripening reaching 10⁶–10⁷cfu g⁻¹in the inner portion of the cheese. From this time onwards, the counts slowly decreased to the end of ripening without disappearing completely.The most abundant species in the milk were Klebsiella oxytoca (36% of the isolated strains), Enterobacter cloacae (24%) and Klebsiella pneumoniae (20%). Escherichia coli, constituted the dominant species from the inner portion of the cheeses at the end of ripening (56% of the isolated strains), followed by Hafnia alvei (44%). However, in the samples of the surface portion of the cheese the dominant species at the end of ripening were K. oxytoca (40%), H. alvei (35%) and E. cloacae (20%).     

Critical effect of oxygen on aroma compound production by Proteus vulgaris

Aroma compound production by Proteus vulgaris was studied in a liquid medium under two culture conditions, static or shaken. By using a dynamic headspace technique coupled with gas chromatography–mass spectrometry, 30 volatile components, aldehydes, alcohols, esters, ketones, sulphur compounds and other miscellaneous compounds were identified. P. vulgaris produced a variety of volatile compounds in different amounts, depending on its culture condition. Sensory analysis has shown that the aroma characteristics of each culture condition were clearly differently perceived. The volatile composition was in agreement with the aroma perception. The culture medium obtained without agitation was characterised by a sulphur/rotten egg note and by a high level of H₂S. The culture medium obtained with agitation was characterised by fruity/fresh and cheese notes and by a high level of alcohols and volatile sulphur compounds. Thus, P. vulgaris could be a useful microorganism to produce various and numerous natural aroma compounds, depending on the culture conditions during fermentation.     

Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

Centrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ～10^1.5 cfu) and nonstarter lactic acid bacteria (by ～10⁸ cfu) and a decrease in the counts of Lactobacillus helveticus (by ～10^2.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese.     

Lipolysis in probiotic and synbiotic cheese: The influence of probiotic bacteria, prebiotic compounds and ripening time on free fatty acid profiles

The influence of probiotic bacteria (Lactobacillus casei-01, Bifidobacterium lactis B94), prebiotic compounds (FOS and inulin) and ripening time (0-60 days) on the free fatty acid (FFA) profile of cheese, with special emphasis on the conjugated linoleic acid (CLA) content, was investigated. After 60 days of ripening, 10⁹-10¹⁰ cfu g⁻¹ cheese were recorded in both probiotic and synbiotic cheeses, despite harsh conditions of low pH values (4.1-5.1) and low moisture content (<30%, w/w). Increases in total FFA and CLA were observed throughout the ripening period, especially in synbiotic cheeses containing FOS and inulin (50:50) inoculated with B. lactis B94. The addition of FOS alone or combined with inulin did not significantly affect probiotic strain growth and viability during the ripening period; however, the advantage of the addition of prebiotic compounds in probiotic cheese manufacture is that it may allow the production of cheeses with improved performance as far as functional CLA compounds are concerned, as well as an improved nutritional quality reflected in a lower atherogenicity index.     

Formation of the aroma of a raw goat milk cheese during maturation analysed by SPME–GC–MS

The volatile profile of the Spanish goat raw milk cheese of the protected designation of origin (PDO) “Queso Ibores” was studied at four stages of maturation (day 1, 30, 60, and 90) by the method of solid-phase micro-extraction–gas chromatography–mass spectrometry (SPME–GC–MS) to determinate the characteristic volatile compounds of this cheese and to know the changes in the volatile profile of this cheese during maturation. According to the PDO, Ibores cheese aroma varies between sweet and mild and it has a strong taste, slightly tart. A total of 64 compounds were detected: 14 acids, 18 alcohols, 13 esters, 6 ketones and 13 compounds which could not be classified in these groups. Carboxylic acids were the most abundant volatile compounds in the headspace of Ibores cheese. Content of volatile compounds was significantly modified (P < 0.05) during ripening. The relative total amounts of acids, esters and ketones increased during the first 60 days of maturation. The most characteristic compounds of Ibores cheese aroma were butanoic, hexanoic and octanoic acids, some alcohols (2-butanol and 2-heptanol), ethyl esters of hexanoic and butanoic acids, some methyl ketones (2-butanone, 2-pentanone and 2-heptanone) and δ-decalactone.     

Olfactometric characterization of aroma active compounds in fermented fish paste in comparison with fish sauce,fermented soy paste and sauce products

Aroma active compounds in commercial fermented fish meat paste product (fish miso), fermented soy paste (soy miso), fish sauce and soy sauces were characterized by using a dynamic headspace method for volatile isolation and GC olfactometry for odor perception. A total of 123 volatile compounds consisting mainly of aldehydes, alcohols, esters, ketones, furans, sulfur and nitrogen-containing compounds, aromatics and acids were consistently identified. A major 16 odor-active compounds were distinguished to contribute as key aroma compounds for the miso and sauce products. Olfactometric and sensory findings clearly differentiated miso products with caramelic, fruity aroma notes, whereas fish sauce products were characterized by ammoniacal, fishy, nutty and cheesy odor note. Soy sauce products, however, were dominated by nutty and cheese aroma. Use of koji for fish miso production was found effective to enhance sweet aroma to the product with a reduction of nutty, meaty and rancid nuance. Principal component analysis employed for statistical interpretations clearly elucidated the relationship among different types of fermented products.     

Isolation of Aroma-Bearing Material from Lactobacillus helveticus Culture and Cheese

In whey permeate supplemented with amino acids and inoculated with Lactobacillus helveticus, methionine was necessary for good aroma development. The aroma of Lactobacillus helveticus cultures could be removed by passing the culture fluid through either acid or basic ion-exchange resins. When the ion-exchange resins were eluted with appropriate volatile acid or base and the eluates were evaporated, the residues slowly generated volatile aroma compounds. Several acetamides were identified in culture extracts. The culture eluates from acid ion-exchange resins were fractionated into a caramelized-burned and a cereal-like component. The burned component was a complex of carbonyls with amino acids, and the burned flavor was attributed to 2,5-dimethyl-4-hydroxy-3[2H] furanone. The cereal component contained a number of volatile amines among which pyr-rolidine had the greatest aroma impact. Various pyrazines, indole, and skatole also were identified. Aqueous cheese extracts yielded aroma-bearing fractions that behaved like the culture aromas on ion-exchange chromatography. The cheese extracts contained aromas with the same gas chromatographic retention times as those in the cultures.     

Dynamic Headspace Analyses of Volatile Compounds of Cheddar and Swiss Cheese during Ripening

The volatile compounds of Cheddar and Swiss cheeses during ripening for 9 wks at 11°and 21°C, respectively, were analyzed by a dynamic headspace analyzer/gas chromatograph every week. The compounds were identified by a combination of retention times and mass spectra. The volatile compounds of Cheddar increased 5.6 and Swiss cheese 15 times as ripening increased from 0 to 9 wks. The amount of volatile compounds of Swiss cheese was 2.6 times greater than that of Cheddar cheese during ripening. The volatile compounds were ketones, alcohols, aldehydes, esters, acids, sulfur compounds, benzenes, and hydrocarbons. Ketones and alcohols accounted for 92% of volatiles from Cheddar cheese and 88% of those from Swiss cheese.     

Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores

This paper reports the technological effects of inoculating Cabrales cheese (a traditional, Spanish, blue-veined cheese) with Penicillium roqueforti spores. Three batches of inoculated Cabrales cheese were manufactured and a number of their microbial and biochemical variables recorded. The results were compared with those obtained for three batches of control cheese made using traditional technology (i.e., adding neither starter cultures nor fungal spores). Although mould and yeast populations grew more quickly in the inoculated cheeses, their normally dominant and representative microbial populations were not affected neither was their gross biochemical composition changed. The variations observed were thought to be caused by the uncontrolled environmental conditions of manufacture and ripening. The development of free amino acids and volatile compounds was significantly increased at 30 days in the inoculated cheeses, although the values for both types of cheese were almost identical at 90 days. The inoculated cheeses obtained higher scores in a hedonistic sensorial evaluation. Thus, inoculation improved the standardization and quality of the cheese.     

Lactococcal aminotransferases AraT and BcaT are key enzymes for the formation of aroma compounds from amino acids in cheese

Amino acid catabolism plays a major role in cheese aroma development. Previously, we showed that the lactococcal aminotransferases AraT and BcaT initiate the conversion of aromatic amino acids, branched-chain amino acids and methionine to aroma compounds. In this study, we evaluated the importance of these two enzymes in the formation of aroma compounds in a cheese model by using single araT and bcaT mutants and a double araT/bcaT mutant. We confirmed that addition of α-ketoglutarate, a co-substrate of aminotransferases, stimulates the conversion of amino acids to aroma compounds in cheese. The results demonstrated that AraT and BcaT are essential for conversion of aromatic and branched-chain amino acids to aroma compounds by Lactococcus lactis in the cheese model and that they also play a major role in the formation of volatile sulphur compounds from methionine. However, another pathway or another aminotransferase appears also to be weakly involved in the formation of these sulphur compounds.     

Key aroma compounds identified in Cheddar cheese with different ripening times by aroma extract dilution analysis,odor activity value,aroma recombination,and omission

To determine the odor-active compounds in Cheddar cheeses with different ripening times (6, 10, and 14 mo), 39 potent odorants of Cheddar cheeses were identified with a flavor dilution factor range between 1 and 512 by aroma extract dilution analysis. To further determine their contribution to the overall aroma profile of Cheddar cheeses, odor activity values of 38 odorants with flavor dilution factors ≥1 were calculated. A Cheddar cheese matrix was developed to determine the concentrations and the odor thresholds of these key aroma compounds. The result of the aroma recombinant experiment prepared by mixing the key aroma compounds in the concentrations in which they occurred in Cheddar cheeses showed that the overall aroma profile of the recombinant sample was very similar to that of Cheddar cheese. The main different compounds in Cheddar cheese with different ripening time were acetic acid, butanoic acid, dimethyl trisulfide, methional, hexanal, (E)-2-nonenal, acetoin, 1-octen-3-one, δ-dodecalactone, furaneol, hexanoic acid, heptanal, and ethyl caproate. This study could provide important information for researching and developing Cheddar cheese–related products.     

Effect of heterofermentative lactic acid bacteria of DL-starters in initial ripening of semi-hard cheese

The role of heterofermentative lactic acid bacteria from dl-starters in ripening of semi-hard cheese was investigated using the strains Leuconostoc pseudomesenteroides PS12 and 1159, Leuconostoc mesenteroides subsp. cremoris T26 and Lactobacillus danicus 13M1. Control cheese was made with starter containing only homofermentative Lactococcus lactis subspecies. Leuc. mesenteroides subsp. cremoris T26 did not grow in cheese and started to decrease in number early, whereas the others grew and remained at a high number throughout the nine-week ripening period. None of the added heterofermentative strains affected proteolysis and total amount of amino acids; however, differences in the composition of amino acids were observed, and caused significant differences in the composition of volatile aroma compounds. Added strains increased the amount of secondary alcohols and mediated decreases in the amount of corresponding methyl ketones, diacetyl and acetoin. Eye formation was only affected by Lb. danicus through stimulation of late gas formation in cheeses.     

Manufacture of Turkish Beyaz cheese added with probiotic strains

The survival of the probiotic strains Lactobacillus fermentum (AB5-18 and AK4-120) and Lactobacillus plantarum (AB16-65 and AC18-82), all derived from human faces, was investigated in Turkish Beyaz cheese production. Three batches of Turkish Beyaz cheese were produced: one with the test probiotic culture mix (P), another with a commercial starter culture mix including Lactoccocus lactis subsp. cremoris, Lactococcus lactis subsp. lactis (C) and the third with equal parts of the commercial starter culture mix and test probiotic culture mix (CP). The cheeses were ripened at 4 °C for 120 days and the viability of cultures was determined monthly. Cheese samples were analyzed for total solids, fat in solids, titratable acidity, pH, salt in total solids, proteolysis, sensory evaluation, aroma compounds and biogenic amines. While initial lactic acid bacteria load in P cheese was 2.7 × 10⁹ at the beginning, it was 7.42 × 10⁷ cfu/g at the end of 120 days of ripening. The results showed that test probiotic culture mix was successfully used in cheese production without adversely affecting the cheese quality during ripening. The chemical composition and sensory quality of P cheeses were also comparable with C cheeses. The present study indicates that probiotic cultures of human origin are feasible for Turkish Beyaz cheese production.     

High-pressure processing decelerates lipolysis and formation of volatile compounds in ovine milk blue-veined cheese

Enzyme-rich cheeses are prone to over-ripening during refrigerated storage. Blue-veined cheeses fall within this category because of the profuse growth of Penicillium roqueforti in their interior, which results in the production of highly active proteinases, lipases, and other enzymes responsible for the formation of a great number of flavor compounds. To control the excessive formation of free fatty acids (FFA) and volatile compounds, blue-veined cheeses were submitted to high-pressure processing (HPP) at 400 or 600 MPa on d 21, 42, or 63 after manufacture. Cheeses were ripened for 30 d at 10°C and 93% relative humidity, followed by 60 d at 5°C, and then held at 3°C until d 360. High-pressure processing influenced the concentrations of acetic acid and short-chain, medium-chain, and long-chain FFA. The effect was dependent on treatment conditions (pressure level and cheese age at the time of treatment). The lowest concentrations of acetic acid and FFA were recorded for cheeses treated at 600 MPa on d 21; these cheeses showed the lowest esterase activity values. Acetic acid and all FFA groups increased during ripening and refrigerated storage. The 102 volatile compounds detected in cheese belonged to 10 chemical groups (5 aldehydes, 12 ketones, 17 alcohols, 12 acids, 35 esters, 9 hydrocarbons, 5 aromatic compounds, 3 nitrogen compounds, 3 terpenes, and 1 sulfur compound). High-pressure processing influenced the levels of 97 individual compounds, whereas 68 individual compounds varied during refrigerated storage. Total concentrations of all groups of volatile compounds were influenced by HPP, but only ketones, acids, esters, and sulfur compounds varied during refrigerated storage. The lowest total concentrations for most groups of volatile compounds were recorded for the cheese pressurized at 600 MPa on d 21. A principal component analysis combining total concentrations of groups of FFA and volatile compounds discriminated cheeses by age and by the pressure level applied to HPP cheeses.     

Effect of mesophilic lactobacilli and enterococci adjunct cultures on the final characteristics of a microfiltered milk Swiss-type cheese

The effect of four associations of adjunct cultures composed of mesophilic lactobacilli and enterococci, either solely or combined, on the microbiological, biochemical and sensory characteristics of Swiss-type cheese made using microfiltered cows’ milk and supplemented with propionibacteria was studied. The global pattern of growth was similar to that generally observed in raw milk cheese and interactions between microflora were highlighted during ripening. Enterococci, which negatively affected the survival of streptococci starters, seemed to play a limited role in the formation of volatile compounds, probably due to their low levels throughout ripening. On the contrary, mesophilic lactobacilli, which affected the evolution of propionibacteria, enterococci and L. delbrueckii subsp. lactis starter counts, modified free amino acid content, production of volatile compounds and organoleptic properties of mature cheese. This population appeared to be of major importance in the formation of cheese flavor as it was positively related to numerous potential flavor compounds such as alcohols and their corresponding esters, acetaldehyde and 4-methyl-4-heptanone. The original mesophilic lactobacilli present in milk could play an important role in the sensorial diversity of raw milk Swiss-type cheeses such as Comte.