The effect of cysteine on production of volatile sulphur compounds by cheese-ripening bacteria

The effect of cysteine on the ability of smear cheese-ripening bacteria (Brevibacterium linens and Arthrobacter spp) to produce volatile sulphur compounds (VSC) from methionine was studied. These bacteria were cultivated in a synthetic medium supplemented with various cysteine concentrations with or without methionine. Cultures with only cysteine showed slightly lower levels of VSC produced and an unpleasant odour like rotten eggs, resulting from hydrogen sulphide production. The levels and profiles of VSC produced with supplemented methionine-cysteine mixtures had strain-dependant behaviours. However, the highest levels of dimethyl disulfide, dimethyl trisulfide and dimethyl tetrasulfide were observed when increasing the cysteine concentration from 0.2 to 1.0 gl(-1) at the same methionine concentration (1.0 gl(-1)). In contrast, production levels of thioesters, especially S-methylthio acetate, were reduced by 50 and 80% under such conditions. An initial sensory approach showed that such an effect could have a strong impact on the global odour of ripened cheeses.     

Short communication: empirical and mechanistic evidence for the role of pyridoxal-5'-phosphate in the generation of methanethiol from methionine

The catabolism of the sulfur-containing AA Met to form flavor-active volatile sulfur compounds (VSC) is an important mechanism in flavor development during cheese maturation. Numerous enzymes catalyzing AA catabolism require the presence of the cofactor pyri-doxal-5′-phosphate (PLP). In fact, reports have shown that some of these reactions can be catalyzed by PLP alone, albeit at a reduced rate. We hypothesized that, as a specific application in cheese flavor reactions, PLP can react directly with free Met to generate a specific VSC, methanethiol (MTH). In this study, the ability of PLP to catalyze MTH generation from Met was examined under “cheeselike” conditions of salt and pH. Methionine and varying concentrations of PLP were incubated in a buffer (pH 5.2 + 4.0% NaCl) analogous to the aqueous phase of aged Cheddar cheese. Samples were analyzed using headspace solid-phase microextraction, and relative concentrations of VSC were determined by gas chromatography-mass spectrometry. Results showed MTH, dimethyl disulfide, and dimethyl trisulfide production when Met and PLP were incubated together at 7°C (cheese-aging temperature). These results indicate that the production of VSC from Met can occur nonenzymatically as catalyzed by free PLP.     

Antimicrobial activity of enterococci strains isolated from white cheese

Twelve strains of enterococci isolated from white cheese samples obtained from different regions of Turkey were screened for their antimicrobial activity against some Gram-positive and Gram-negative food-borne pathogens and contaminating bacteria, also against themselves and some other lactic acid bacteria (LAB), including some species of lactococci, lactobacilli and Leuconostoc . Antimicrobial activity was confirmed for all 12 strains, including six Enterococcus faecium , two Enterococcus faecalis and four Enterococcus durans . Antimicrobial agents produced by these enterococci exhibited a spectrum of activity, which is mainly directed against food-borne pathogens, specially Listeria monocytogenes and Bacillus cereus and some other Bacillus spp. and also some enterococci. Tested LAB was insensitive to inhibitory agents produced by them.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Technological characterization of the natural lactic acid bacteria of artisanal Turkish White Pickled cheese

The aim of this study was to characterize the lactic acid bacteria (LAB) isolated from White Pickled cheeses produced with traditional methods; and to improve the quality of cheesemaking with a selection of bacterial cultures from artisanal White cheeses. LAB were isolated and identified from 30 White Pickled cheese samples collected from various cities in Turkey. Also, the numbers of several microbial groups (total aerobic mesophilic bacteria, LAB, enterococci, coliforms, moulds and yeasts) of cheese samples were enumerated. Lactobacilli, lactococci and enterococci were the most abundant microbial groups. The numbers of Enterococcus and Lactobacillus isolates were higher than those of the other LAB. Enterococcus faecalis (24.43%), Enterococcus faecium (17.61%) and Lactobacillus fermentum (19.88%) isolates were the most frequently isolated species. Lactococcus strains showed the highest acidifying activity, followed by Enterococcus and Lactobacillus strains. Proteolytic activity of Enterococcus faecalis strains was higher than that of the other enterococci species, except Enterococcus avium strains. Within lactobacilli strains, the highest mean proteolytic activity was that of Lactobacillus bifermentans, Lactobacillus brevis and Lactobacillus casei strains.     

Relationships between flavour and microbiological profiles in Serra da Estrela cheese throughout ripening

Cheeses manufactured in certified dairies in the Portuguese region of Serra da Estrela, using refrigerated and non-refrigerated raw sheep milk, were quantitatively evaluated in terms of indigenous microflora and volatile compounds during a ripening period of up to 6 mo. Viable counts were obtained for lactococci, lactobacilli, leuconostoc, enterococci, yeasts, Enterobacteriaceae and staphylococci; analyses of volatiles were performed by chromatography after solid-phase micro-extraction. Treatment of all analytical data produced by principal component analysis revealed correlations between the major microbial groups present in cheese and patterns of volatiles generated. End products resulting from the degradation of sugars, free amino acids and glycerides constituted the predominant volatiles of Serra da Estrela cheeses. Among volatile, short-chain carboxylic acids detected were acetic, propionic, iso-butyric and iso-valeric acids; these compounds are known to be breakdown products of Gly, Ala and Ser, of Thr, of Val, and of Ile, respectively, following oxidative deamination. Semi-volatile fatty acids and their corresponding ethyl esters appeared in the cheese, probably as a result of the activity of lipases produced by yeasts and Enterobacteriaceae. These ethyl esters, which are responsible for fruity flavours, were especially pronounced in cheeses manufactured from refrigerated milk.     

Flavour sulphides are produced from methionine by two different pathways by Geotrichum candidum

We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5'-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography-mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography-mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.     

Identification of Lactic Acid Bacteria Isolated from Tulum Cheese During Ripening Period

The fresh Tulum cheese was manufactured and then ripened at 10 ± 2°C for 3 months. Isolation and identification of lactic acid bacteria (LAB) was carried out during the ripening period of 90 days. Lactic acid bacteria were isolated from PCA, M17, and MRS agar. The strains isolated were identified using morphology, colony pigmentation, production of carbon dioxide from glucose, growth at 4 and 40°C, salt tolerance, starch hydrolysis, and sugar fermentation with the API system methods. A total of 253 strains isolated during the storage. Identifying strains belonged to genera of the lactobacilli (133), pediococci (44), enterococci (29), leuconostocs (27), and lactococci (8). 12 of 253 strains were not also identified. Lactobacilli (75.2%) were frequently determined on MRS medium. In the results of this article, lactobacilli were found at high frequencies, while enterococci, lactococci, leuconostocs, and pediococci were found at low frequencies in Tulum cheese. Lactobacilli increased during the ripening period, but the others did not change a significant amount.     

Screening of lactic acid bacteria for antimicrobial properties from mayonnaise-based products and raw materials

A collection of lactic acid bacteria isolated from both mayonnaise-based products and raw materials used to manufacture them was tested for antimicrobial activity. Out of 144 strains (97 lactobacilli, 23 lactococci and 24 enterococci) only three supernatants of Enterococcus spp. strains (EN3, EN14 and EN15) exhibited activity against lactobacilli and lactococci. The supernatant of the strain EN3 exclusively inhibited the growth of Bacillus cereus and Listeria monocytogenes. Enterococcus strains EN3, EN14 and EN15 produced thermostable bacteriocins, which had antibacterial activity.     

Methionine Metabolism: Major Pathways and Enzymes Involved and Strategies for Control and Diversification of Volatile Sulfur Compounds in Cheese

For economical reasons and to accommodate current market trends, cheese manufacturers and product developers are increasingly interested in controlling cheese flavor formation and developing new flavors. Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to their typical flavors. Thus, the control of VSCs formation offers considerable potential for industrial applications. This paper gives an overview of the main VSCs found in cheese, along with the major pathways and key enzymes leading to the formation of methanethiol from methionine, which is subsequently converted into other sulfur-bearing compounds. As these compounds arise primarily from methionine, the metabolism of this amino acid and its regulation is presented. Attention is focused in the enzymatic potential of lactic acid bacteria (LAB) that are widely used as starter and adjunct cultures in cheese-making. In view of industrial applications, different strategies such as the enhancement of the abilities of LAB to produce high amounts and diversity of VSCs are highlighted as the principal future research trend.     

Key enzymes involved in methionine catabolism by cheese lactic acid bacteria

Cheese microbiota and their enzymatic conversion of l-methionine to volatile sulphur compounds (VSCs) play an important role in aroma formation during cheese ripening. Here, lactic acid bacteria (LAB) strains isolated from raw goats' milk cheeses were screened for the major enzymes critical to the formation of VSCs from l-methionine. A large natural biodiversity in enzyme capabilities and high inter- and intra-species variability was found among the LAB isolates investigated. From those isolates tested, lactococci displayed higher C–S lyase specificities towards the sulphur-containing compounds examined than did Lactobacillus and Leuconostoc, in some cases generating higher levels of VSCs than B. linens, known to be an efficient producer of methanethiol (MTL) and related VSCs. Moreover, these differences in C–S lyase activities (determined spectrophotometrically by measuring the formation of free thiol groups) were shown to correspond with the enzymatic potential of the isolates as determined by visualization of enzymatic activities. This technique could therefore prove valuable for the detection and preliminary characterization of C–S lyase activities among LAB isolates. Lactococci were also found to possess higher aminotransferase activities than lactobacilli and leuconostocs, while glutamate dehydrogenase activities were observed to be highest among Leuconostoc and Lactobacillus spp. Meanwhile, α-keto acid decarboxylase activities were highly variable and were measurable in only a limited number of isolates, mainly lactobacilli. From these data, combining indigenous isolates showing high VSCs-producing capabilities with those that facilitate the completion of the metabolic pathway responsible for degrading l-methionine into volatile compounds may provide an efficient approach to enhance cheese aroma development.     

Proteolysis in Cheese Curd as Affected by Subcellular Fractions from Lactococcus, Lactobacillus and Propionibacterium

Intracellular fraction (IF) and cell wall-associated fraction (CWAF) from lactococci, lactobacilli and propionibacteria were added to cheese curd slurries according to a mixture design. Response surface and estimation of Scheffe's polynomial revealed synergistic interaction effects between lactococci and lactobacilli on secondary proteolysis determined by an o-phthaldialdehyde assay. IFs from propionibacteria had a lower proteolytic activity than those from lactococci and lactobacilli used. Principal component analysis of capillary electropherograms of the trichloroacetic acid-soluble fraction, discriminated between slurries treated with IFs and CWAFs. It also showed an effect of proteolytic enzymes from the IF of propionibacteria together with IFs from lactococci and/or lactobacilli, on small changes in overall peptide profile.     

Impedance measurement of growth of lactic acid bacteria in the presence of nisin and lysozyme

Effect of nisin (0.500 g/l), lysozyme (0.035 g/l) and the mix of nisin (0.500 g/l) and lysozyme (0.035 g/l) against two nisin-producing lactococci strains, three non-nisin-producing lactococci strains and four lactobacilli strains with antimicrobial activity was determined by the impedimetric method. None of the tested lactococci and lactobacilli strains were inhibited by lysozyme at concentration of 0.035 g/l. Only the nisin-producing lactococci strains were resistant to nisin at 0.500 g/l, the other lactococci and lactobacilli were inhibited by nisin at 0.500 g/l intensively. The mix of lysozyme and nisin displayed a comparable effect to the nisin addition against the used lactococci strains and was very variable depending on the strain of tested lactobacilli. The impedimetric method was shown to be convenient for screening the growth abilities of lactococci and lactobacilli in the presence of antimicrobial components.     

Changes in microbial flora during manufacture of a traditional fermented milk from ewe's milk

Changes in the microbial flora during the manufacture of a sour milk from raw ewe's milk were studied. Mean log counts of lactic acid bacteria, lactobacilli and streptococci (respective values in milk 4.55, 3.19 and 5.56) increased significantly (P<0.05) to 8.46, 6.98 and 8.46, respectively, at 5 days and at the same time the pH decreased from 6.58 in milk to 4.71 in the final product. Levels of total counts, coliforms and psychrotrophs also increased to 10⁸, 10⁷and 10⁶cfu/ml, respectively. Halotolerants and yeasts were counted at quite low numbers throughout manufacture.Isolates of lactic acid bacteria were classified as lactococci, lactobacilli, leuconostocs and enterococci. Enterococci predominated (58.9% of the isolates) over the other lactic acid bacteria after 1 day meanwhile leuconostocs became predominant (46.9%) after 5 days, followed by lactobacilli (29.7%), lactococci (17.2%) and enterococci (6.2%).Lactobacillus plantarumandLeuconostoc mesenteroidessub-spp.cremoriswere the species most frequently isolated. Enzyme activities (API-ZYM tests) of lactococci and leuconostocs were low, in general, but activities ofLb. plantarumstrains were considerably higher. ThreeLc. lactissub-spp.lactisstrains produced diacetyl, four of them formed acetoin, and one was characterized as good active, concerning acidification ability.Results presented here suggest the necessity of using a starter culture, consisting of selected strains of lactococci, leuconostocs and possibly lactobacilli, and pasteurized milk for the production of a high quality product.     

Microbiological changes throughout ripening of goat cheese made from raw,pasteurized and high-pressure-treated milk

The bacteriological quality during ripening of raw (RA), pasteurized (PA; 72°C, 15 s) and pressure-treated (PR; 500 MPa, 20°C, 15 min) goat milk assessed by enumeration of total bacteria, psychrotrophic bacteria, Enterobacteriaceae, lactobacilli, enterococci, Micrococcaceae and lactococci was evaluated. The high pressure treatment applied was as efficient as pasteurization in reducing the bacterial population of milk. Experimental cheeses were made from RA, PA and PR milks to study the microbial population during ripening. Lactobacilli and lactococci were the predominant microbiota present during ripening in all the cheeses. There were no differences in numbers of starter bacteria during ripening. However, lactobacilli counts for RA milk cheese were significantly higher than for PA and PR cheeses in all the ripening stages studied. Micrococcaceae and enterococci remained at a secondary level, and no differences were observed between cheeses at the end of ripening. On the other hand, the number of Enterobacteriaceae decreased during ripening, but faster in PR milk cheese than in PA and RA milk cheeses. The results of this study suggest that goat cheese made from PR milk had similar microbiological characteristics to PA milk cheeses.     

Inactivation of lactococcal aromatic aminotransferase prevents the formation of floral aroma compounds from aromatic amino acids in semi-hard cheese

The enzymatic conversion of aromatic amino acids to aroma compounds plays a role in the formation of an undesirable floral aroma in Cheddar-like cheeses. In lactococci, the first step of aromatic amino acid degradation is a transamination, catalysed by an aromatic aminotransferase (AraT). We observed previously that in vitro, araT inactivation prevented degradation of aromatic amino acids and decreased degradation of Met and Leu. In this study we evaluated the effect of araT inactivation in Lactococcus lactis on flavour development in St. Paulin-type cheese. The degradation of amino acids was monitored by using radiolabelled amino acids and the volatile compounds formed were analysed by GC-MS. The development of cheese odour was also evaluated by sniffing. We confirmed that the availability of an -ketoacid acceptor for transamination is the first limiting factor for amino acid conversion to aroma compounds in cheese. In the presence of -ketoglutarate, araT inactivation greatly prevented formation of floral aroma compounds from aromatic amino acids while it did not affect the formation of volatile aroma compounds from branched-chain amino acids and methionine. However, the sensory analysis by sniffing did not reveal any significant effect of the gene inactivation although the odour of cheese made with the mutant tended to be less floral than that of cheese made with the wild type strain.     

天祝牧区牦牛乳酸奶中优良乳酸菌的分离及筛选

从天祝牧区采集的22 份牦牛乳酸奶样品中分离得到96 株乳酸菌。通过初筛：包括遗传稳定性、凝乳时间、滴定酸度、保水率指标的测定及感官评价,得到12 株凝乳时间短、遗传性状稳定的乳酸菌。通过复筛：包括酸化能力、后酸化能力、产乙醛和双乙酰能力、蛋白质分解能力、冷藏期间活菌数、耐受不良环境能力的测定,最终筛选出性能优良的4 株杆菌和2 株球菌。     

The biodiversity and evolution of lactic flora during ripening of the Iranian semisoft Lighvan cheese

Ninety‐five isolated strains of Lactic acid bacteria (LAB) were identified from Lighvan cheese. The LAB evolution showed the dominance of lactococci and lactobacilli in the first stage and substitution of these genera by enterococci at the end of ripening. The most predominant strains were Enterococcus faecium (22.44%), Lactococcus lactis ssp. lactis (20.4%), Lactobacillus plantarum (18.36%) and E. faecalis (14.28%), respectively. Eleven and 51 different carbohydrate fermentation profiles were observed according to API 20 STREP and API 50 CH, respectively. API 20 STREP dendogram showed identical fermentation profiles of some E. faecalis and E. faecium strains, indicating that these strains might be well adapted to the whole cheese manufacture.     

Volatile compounds produced in cheese by Enterobacteriaceae strains of dairy origin

The formation of volatile compounds in fresh cheese by 10 Enterobacteriaceae strains of dairy origin (4 Hafnia alvei, 2 Serratia liquefaciens, 1 Enterobacter cloacae, 1 Enterobacter sakazakii, and 2 Escherichia coli strains) was investigated. Small cheeses were made from pasteurized cow's milk separately inoculated with 1-3 x 10(3) CFU/ml of each of the Enterobacteriaceae strains, with glucono-8-lactone added to achieve a pH value of 5.2 in the curds. All strains reached counts close to 10(8) CFU/g in 1-day-old cheeses and survived well from day 1 to day 8. Cheeses were analyzed for volatile compounds by gas chromatography-mass spectroscopy, after extraction by dynamic headspace using a purge and trap apparatus. Sixty-one volatile compounds were determined in cheeses, 31 of which were further investigated. Significant increases of aldehydes, sulfur compounds, and aromatic compounds were recorded from 2-h curd to 1-day-old cheese, and of ketones, alcohols, and acids from 2-h curd to 8-day-old cheese. Acetaldehyde, 2-methyl propanal, and 3-methyl butanal predominated among aldehydes; 2,3-butanedione, 2,3-pentanedione, and 3-hydroxy 2-butanone among ketones; ethanol, 2-methyl propanol, and 3-methyl butanol among alcohols; and ethyl acetate among esters. Hierarchical cluster analysis of strains using the data of 31 volatile compounds separated clearly the strain of E. sakazakii, which produced high amounts of volatile compounds, from the other Enterobacteriaceae strains.