Characterisation of the microflora during ripening of a Norwegian semi-hard cheese with adjunct culture of propionic acid bacteria

The microflora of a semi-hard, washed curd, Norwegian cheese with an added adjunct culture of propionic acid bacteria (PAB) was investigated throughout ripening by phenotypic and physiological tests, API test and 16S rRNA sequencing. Cheeses were made at two commercial Norwegian dairies using different milk treatments (pasteurisation versus microfiltration plus pasteurisation) and the same type of starter cultures. Microflora in the cheese varied according to different plant site, milk treatment, and ripening time. PAB dominated the microflora throughout the ripening process. Leuconostoc spp., most probably from the starter, dominated among the isolates from the cheese using microfiltered and pasteurised milk; however, after 40 weeks of ripening non-starter lactic acid bacteria specie Lactobacillus casei/paracasei and Leuconostoc spp. dominated at the dairy using pasteurised milk. Cheese made at the two plants on two subsequent days showed almost identical microflora throughout ripening.     

Effect of the inclusion of artichoke silage in the ration of lactating ewes on the properties of milk and cheese characteristics during ripening

The effect of including artichoke silage in the rations of dairy ewes on milk characteristics and biochemical changes of ripened cheeses was evaluated. Four groups of lactating ewes were fed rations containing 0, 10, 20, or 30% artichoke silage on a dry matter basis. Bulk milk samples were collected 3 times during the feeding period, and semi-hard cheeses were manufactured and sampled during ripening. Milk composition and cheese yield were not affected by diet. Inclusion of 20 and 30% artichoke silage reduced the firmness of the curds at a level only detected by the Gelograph (Gelograph-NT, Gel-Instrumente, Thalwil, Switzerland) probe. Inclusion of artichoke silage in ewes’ diet decreased fat and total free fatty acids content of these cheeses and increased total free amino acids content. Despite the effect of diet on cheese ripening characteristics, the overall sensory scores for cheeses corresponding to artichoke silage diets were statistically higher than those for the control cheeses.     

Microbiological,chemical, textural and sensory characteristics of Hispánico cheese manufactured using frozen ovine milk curds scalded at different temperatures

Hispánico cheese is a semi-hard variety, manufactured in Spain from a mixture of pasteurized bovine and ovine milk. To study one strategy for overcoming a seasonal shortage of ovine milk in summer and autumn, curds made from ovine milk, scalded at 32, 35 or 38 °C, were pressed for 30 min and frozen at ?24 °C for 4 months. After thawing, they were added to fresh bovine milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized bovine and ovine milk in the same (80:20) proportion. No significant effect of the addition of frozen ovine milk curd or scalding temperature was found for lactic acid bacteria counts, dry matter content, hydrophilic and hydrophobic peptides, 45 out of 65 volatile compounds, texture, and sensory characteristics throughout a 60-day ripening period. Differences between cheeses, of low magnitude and little practical significance, were found for pH value, aminopeptidase activity, proteolysis, free amino acids, free fatty acids, and the remaining 20 volatile compounds. Thus, the addition of frozen ovine milk curd to fresh bovine milk curd does not alter the main physicochemical and sensory characteristics of Hispánico cheese.     

乳清添加对马苏里拉奶酪成熟过程中风味物质及其品质的影响

该研究以未添加乳清的马苏里拉奶酪为对照,考察乳清对马苏里拉奶酪成熟期内（0 d、30 d、60 d、90 d）乳酸菌活菌数、油脂析出性、挥发性风味物质及氨基酸含量的影响,并对挥发性风味物质与氨基酸进行相关性分析。结果表明,在奶酪成熟期内,两种奶酪的活菌数均呈先增加后减少的趋势,油脂析出性均增加,且乳清奶酪均高于对照奶酪;两种奶酪的挥发性风味物质存在差异,从2种奶酪中共检测到61种挥发性风味物质,其中对照奶酪中共检出43种,乳清奶酪中共检出55种。乳清奶酪中的醇、酸、醛、酮、酯类物质的含量及种类均高于对照。两种奶酪成熟期间均分别检测到15种游离氨基酸,乳清奶酪成熟90 d时的氨基酸含量（848.92 mg/kg）高于对照奶酪（663.44 mg/kg）。除精氨酸、丝氨酸、酪氨酸、天冬氨酸外,其余氨基酸对20种挥发性风味物质具有积极作用。综上,乳清添加可提升马苏里拉奶酪的风味。     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.     

不同包装方式对半硬质干酪成熟期间蛋白质降解的影响

对真空包装和涂蜡包装的半硬质干酪成熟过程中蛋白降解进行了研究。结果表明:2种包装的干酪在成熟过程中pH 4.6SN含量和12%TCA-SN含量都随着时间的延长逐渐增大,且2组数据之间差异显著(P<0.05);2种包装的干酪中游离氨基酸总量随成熟时间的延长而逐渐增加,各种氨基酸含量变化的显著性不同;SDS-PAGE电泳图谱显示2种干酪在成熟期内蛋白质都发生了明显的降解,且涂蜡包装的干酪蛋白降解程度较真空包装的深,在成熟45 d后较为明显。     

Influence of the starter culture on the microbiological and sensory characteristics of ewe's cheese

Changes which take place in the sensory characteristics of cheeses during ripening are influenced by different factors, involving rennet, starter culture and adventitious contamination of the cheese by non-starter lactic acid bacteria. The objective of this work was to study the influence of the starter on sensory and microbiological ewe's cheese properties during ripening time. Four batches (two with starter added and two without) were manufactured. Milk and cheeses at different stages of ripening were analysed. Cheeses manufactured without adding starter showed a significantly higher level of mesophilic aerobic microflora, lactobacilli, facultatively heterofermentative lactobacilli and enterococci (indigenous microflora) than cheeses manufactured with starter. This study has also shown that adding or not adding starter affects the flavour profile of the cheese. Cheeses with starter added showed greater intensity of the following attributes: refreshing, astringent, sweet; and received lower scores on bitterness. With respect to texture, the said cheeses develop a more homogenous texture and greater elasticity throughout ripening.     

Texture, proteolysis and viable lactic acid bacteria in commercial cheddar cheeses treated with high pressure

High pressure processing was investigated for controlling Cheddar cheese ripening. One-month-or 4-month-old Cheddar cheeses were subjected to pressures ranging from 200 to 800 MPa for 5 min at 25 C. The number of viable Lactococcus lactis (starter) and Lactobacillus (nonstarter) cells decreased as pressure increased. Subsequent storage of the control and pressure-treated cheeses at 10 degrees C caused viable cell counts to change in some cases. Free amino acid content was monitored as an indicator of proteolysis. Cheeses treated with pressures > or = 400 MPa evolved free amino acids at significantly lower rates than the control. No acceleration in free amino acid development was observed at lower pressures. Pressure treatment did not accelerate the rate of textural breakdown compared with the non-pressure treated control. On the contrary, pressure treatment at 800 MPa reduced the time-dependent texture changes. Results indicate that high pressure may be useful in arresting Cheddar cheese ripening.     

Biochemical patterns in ovine cheese: Influence of probiotic strains

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of α_S1-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of α-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on the basis of highest content of health-promoting molecules. The metabolic activity of the cheese microflora was also monitored by measuring acetic, lactic, and citric acids during cheese ripening. Cheese acceptability was expressed for color, smell, taste, and texture perceived during cheese consumption. Use of probiotics in trial cheeses did not adversely affect preference or acceptability; in fact, panelists scored probiotic cheeses higher in preference over traditional cheese, albeit not significantly.     

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening

Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe's milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses.     

The effect of three different ripening/storage conditions on the distribution of selected parameters in individual parts of Dutch‐type cheese

The aim of the study was (i) to detect changes of dry matter, NaCl and twenty‐two free amino acids contents, pH and levels of selected microorganisms in four layers of cheese (from edge to core) during ripening and storage period and (ii) to describe the changes of the above‐mentioned parameters caused by early relocation of cheese from optimum ripening conditions to refrigeration temperatures. The number of mesophilic aerobic and facultative anaerobic bacteria and lactic acid bacteria differed significantly (P < 0.05) during the experiment dependent on the analysed layer and ripening/storage conditions. The free amino acid content differed significantly in individual analysed layers of cheese and also according to individual ripening/storage conditions. The highest content of free amino acids was found in samples stored at optimal ripening temperatures. Cheese hardness was also analysed and the lowest one was detected in samples ripened under optimal temperatures for the whole period. Early release of cheeses into storage rooms with lower temperature significantly affected properties of these products.     

Effects of heating milk and accelerating ripening of low fat Ras cheese on biogenic amines and free amino acids development

Two experiments were conducted to study the effects of heating milk and, consequently, incorporation of whey proteins into cheese curd, fat content, accelerating cheese ripening by attenuated lactobacilli, species of bacteria and method of attenuation on formation of biogenic amines and liberation of free amino acids. Total biogenic amines and total free amino acids increased as ripening period progressed in all cheese treatments. Total biogenic amines and total free amino acids decreased as the fat content was decreased. Heating of 3% fat milk up to 70°C caused a significant (p<0.05) increase in total biogenic amines and total free amino acid concentrations, while raising the temperature of heat treatment up to 75 and 80°C decreased them. However, heating of 2% fat milk up to 75°C caused a definite (p<0.05) increase in total biogenic arnines and total free amino acids; conversely raising heat treatment temperature to 80°C decreased them. These results indicate that there is a positive correlation between total biogenic amines and total free amino acids; moisture and salt contents affected the formation of biogenic amines, while incorporation of whey proteins had no significant effect on biogenic amines development. In a second experiment, addition of attenuated lactobacilli as adjunct bacteria caused a pronounced (p<0.05) increase in free amino acids and biogenic amines. Addition of either freeze- or heat-shocked Lactobacillus helviticus was more effective in promoting the build-up of biogenic amines and free amino acids in the resultant cheeses than in cheeses made with addition of freeze- or heat-shocked Lactobacillus casei, respectively. There was a positive correlation between free amino acids and biogenic amine contents. The type of bacteria and prolongation of ripening period significantly affected the development of biogenic amines. Tyramine was the highest biogenic amine followed by histamine, while spermidine was the lowest.     

Changes in organic acids during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk

Organic acids of cheeses made from raw (RA), pasteurized (PA; 72°C, 15 s) or pressure-treated (PR; 500 MPa, 15 min, 20°C) goats’ milk were qualitatively and quantitatively assessed during ripening. Nine organic acids (citric, pyruvic, malic, lactic, formic, acetic, uric, propionic and butyric) were analysed in each sample by HPLC.Milk treatment did not affect the total organic acids content of 1-day-old cheeses, which increased steadily from day 1 to day 60. At the end of ripening, RA and PR milk cheeses both exhibited higher concentration of organic acids than in those made from PA milk.Lactic acid was found in higher concentration in PR milk cheese from 30 days of ripening. The RA milk cheese, that showed the highest nonstarter lactic acid bacteria counts, were characterized by an elevated amount of propionic and acetic acids. These cheeses also were negatively correlated with both pyruvic and citric acid contents. The PA milk cheese showed a high level of malic acid, and was clearly differentiate from RA and PR milk cheeses by its low level of butyric acid.     

不同菌种大豆干酪后熟过程的研究

研究了3种不同大豆干酪后熟过程的变化情况。结果表明,L.rhamnosus+S.carnosus大豆干酪具有最低的pH和最高的酸度,同时具有最高的氨基氮含量和最高的活菌数。游离氨基酸分析表明,L.rhamnosus+S.carnosus大豆干酪中的游离氨基酸总量最高;L.rhamnosus大豆干酪降解产生的游离氨基酸以谷氨酸和精氨酸为主,S.carnosus大豆干酪和L.rhamnosus+S.carnosus大豆干酪降解产生的游离氨基酸则以色氨酸为主。脂肪酸分析表明,L.rhamnosus+S.carnosus大豆干酪C20以上的长链脂肪酸得到更加充分的降解。     

Effect of artisanal kid rennet paste on lipolysis in semi-hard goat cheese

This study examined the use of hygienised kid rennet pastes in model cheese systems and also in goat milk semi-hard cheeses to promote lipolysis. The results obtained indicated that the use of rennet paste caused greater lipolysis and increased, mostly, the short-chain free fatty acid (FFA) content. The model systems made with whole goat’s milk using rennet paste and commercial rennet mixture exhibited a higher FFA content than did the rennet paste-free controls (31,600 vs. 25,600 μmol/kg cheese). For the pilot cheeses made with bovine rennet and rennet paste mixture, the increase in FFA level after 45 days of ripening compared with the cheeses prepared only with commercial rennet was as much as 6600 (μmol/kg cheese) and the increase in the butyric acid content was also 1650 (μmol/kg cheese). Moreover, after 15 days of ripening, industrially prepared cheeses made with rennet paste exhibited greater levels of FFA than did the cheeses made with commercial rennet (11,500 μmol/kg at 45 days of ripening). Their flavour was stronger and the organoleptic characteristics were better accepted, which implies less ripening time for commercial cheese manufacture.     

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are:

• a partial elimination of the milk microorganisms which may grow in cheese during ripening,

• a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization.,

• a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria).

From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae, enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of _s1- and β-caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.     

Changes of free amino acid content of Teleme cheese made with different types of milk and culture

The evolution of concentration of free amino acids in Teleme cheese made from sheep, goat or cow milk, using a thermophilic, mesophilic or a mixture of a thermophilic, a mesophilic culture throughout ripening was studied. The total free amino acid (TFAA) content increased at all stages of ripening, regardless of the milk and culture used. In general, the TFAA content was higher in cheeses made from cow’s milk than that of the cheeses made from ewe’s milk; cheese from goat’s milk ranged over intermediate levels. Also, higher concentrations of TFAA were found in cheeses made with the thermophilic than with the mesophilic culture. Cheeses made with the mixture of thermophilic–mesophilic culture ranged over intermediate levels. The results of this study have shown that Leu, Glu, Phe, Val and Lys were the major FAA of Teleme cheese at all stages of ripening, regardless of the type of milk and culture used.     

Proteolytic and lipolytic changes during the ripening of León raw cow's milk cheese, a Spanish traditional variety

Proteolytic and lipolytic changes were studied throughout ripening of five batches of León cow's milk cheese, a traditional variety made in the north of Spain. Total soluble nitrogen, non-protein nitrogen, oligopeptides nitrogen, amino nitrogen and ammonia nitrogen fractions increased slightly during the ripening process. The final values of these nitrogen fractions indicate that this cheese undergoes a very slight proteolysis as much in extent as in depth. This weak protein degradation is corroborated when the caseins and their degradation products were quantified by electrophoresis. β-Casein stayed practically intact throughout the ripening process and only 10% of α_s-casein became degraded. The content of total free amino acids increased progressively but in a slightly increased way during ripening, reaching final average values of 592 mg (100 g)⁻¹ of total solids. The most abundant free amino acid at the end of ripening was lysine, followed by leucine, glutamic acid, tryptophan, valine and phenylalanine. The acidity index of the fat values increased during ripening by a factor of 4.39. The final values of this parameter are in the range of those observed in other cow's milk cheeses ripened by bacteria. The content in total free fatty acids underwent an increase throughout ripening reaching final average values of 6669 ppm. The most abundant free fatty acid at the end of ripening was oleic acid followed by butyric and palmitic acids. The high content of short-chain fatty acids is outstanding, specially that of butyric acid.     

Evolution of Free Amino Acids during the Ripening of Cheddar Cheese Containing Added Lactobacilli Strains

Cheddar cheese was produced with different lactobacilli strains added to accelerate ripening. The concentration of proteolytic products was determined as free amino acids in the water-soluble fraction at two, four, seven and nine months of aging and at two different maturation temperatures (6°C, 15°C). All amino acids increased during ripening and were higher in the Lactobacillus- added cheeses than in the control cheese, and higher in cheeses ripened at 15°C than at 6°C. Glutamic acid, leucine, phenylalanine, valine and lysine were generally in higher proportion in all cheeses. The cheeses with added L. casei-casei L2A were classified as having a “strong Cheddar cheese” flavor after only seven months of ripening at 6°C.