Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.     

Proteolysis during ripening of Manchego cheese made from raw or pasteurized ewes' milk. Seasonal variation

Changes in nitrogen compounds during ripening of 40 batches of Manchego cheese made from raw milk (24 batches) or pasteurized milk (16 batches) at five different dairies throughout the year were investigated. After ripening for six months, degradation of p-kappa- and beta-caseins was more intense in raw milk cheese and degradation of alpha(s2)-casein in pasteurized milk cheese. Milk pasteurization had no significant effect on breakdown of alpha(s1)-casein. Hydrophobic peptide content did not differ between raw and pasteurized milk cheese, whereas hydrophilic peptide content was higher in raw milk cheese. There were no significant differences between seasons for residual caseins, but hydrophobic peptides were at a higher level in cheese made in autumn and winter and hydrophilic peptides in cheese made in winter and spring. Raw milk cheese had a higher content of total free amino acids and of most individual free amino acids than pasteurized milk cheese. The relative percentages of the individual free amino acids were significantly different for raw milk and pasteurized milk cheeses. The relative percentages of Lys and lie increased, while those of Val, Leu and Phe decreased during ripening. There were also seasonal variations within the relative percentages of free amino acids. In raw milk cheeses, Asp and Cys were relatively more abundant in those made in autumn, Glu and Arg in cheeses made in winter, and Lys and Ile in cheeses made in spring and summer. Biogenic amines were detected only in raw milk cheese, with the highest levels of histamine, tryptamine and tyramine in cheeses made in spring, winter and spring, respectively.     

Early PCR detection of tyramine-producing bacteria during cheese production

Biogenic amines (BA) are toxic substances that appear in foods and beverages. Tyramine is the most abundant BA in cheeses. A PCR method was developed to detect the presence of tyramine-producing bacteria during cheese manufacture and ripening. Six different batches of a farmhouse blue cheese were analysed by PCR. Tyramine concentrations were also determined by HPLC. The PCR method was able to anticipate tyramine accumulation in the cheeses; the presence of tyramine-producing microorganisms in the early stages of manufacture correlated well with a high concentration of BA in mature cheese samples.     

Biogenic Amines Content of Four Types of “Pecorino” Cheese Manufactured in Tuscany

Biogenic amines content of four types of Tuscan ewes’ milk “pecorino” cheese was evaluated using HPLC-UV analysis. All cheeses were manufactured in the same dairy factory with different combinations of milk (raw or pasteurized) and type of ripening. Total biogenic amines and tyramine levels of a raw milk “pecorino” ripened five months, partly in a traditional cave, were significantly higher than those of a pasteurized milk “pecorino” with a similar ripening; and of a two months raw milk “pecorino” ripened in the dairy plant. No statistical significant difference was found when comparing total biogenic amines and tyramine contents of the same five month ripened raw milk “pecorino” with a pasteurized milk “pecorino” ripened six months, partly in a traditional “fossa.” In raw milk cave-ripened and “fossa”-ripened cheeses, total biogenic amines exceeded 1000 mg/kg. In cheeses manufactured with raw milk and/or in particular ripening environments, specific hygienic cares are needed to limit biogenic amines formation.     

Influence of pasteurised milk, raw milk and different ripening cultures on biogenic amine concentrations in semi-soft cheeses during ripening

In semi-soft cheeses, produced with pasteurised milk, raw milk and different starter cultures, the concentrations of cadaverine, histamine, phenylethylamine, putrescine and tyramine were investigated. The cultures (pasteurised milk cultures, raw milk cultures and starter cultures) strongly influenced the biogenic amine concentrations in the cheeses ripened for 5 months. Two cheeses made with identical pasteurised milk, but different ripening cultures, differed greatly in their total biogenic amine concentrations (51 vs 371?mg/kg). In general, the biogenic amine concentrations increased markedly between month 2 and month 3 of cheese ripening. The high content of enterococci and Enterobacteriaceae yielded the biogenic amine concentrations. In contrast, Lactobacilli did not seem to be important. However, unspecified bacteria have to be considered, since cheeses with comparable microbiological profiles differed enormously in their biogenic amine concentrations. Semi-soft cheeses produced from pasteurised milk showed remarkably lower total biogenic amine concentrations compared to semi-soft cheeses produced from raw milk (51–1096?mg/kg vs 1011–3133?mg/kg, depending also on the ripening cultures). The highest total biogenic amine concentration (4817?mg/kg) was detected in a cheese produced from raw milk that had been stored for 36?h. In this cheese, the concentrations of cadaverine, phenylethylamine, putrescine and tyramine were higher than in all other cheeses. The highest histamine concentration was found to be in another raw milk cheese (573?mg/kg).     

Factors affecting consumers' preferences for and purchasing decisions regarding pasteurized and raw milk specialty cheeses

Eight hundred ninety consumers at a local food festival were surveyed about their specialty cheese purchasing behavior and asked to taste and rate, through nonforced choice preference, 1 of 4 cheese pairs (Cheddar and Gouda) made from pasteurized and raw milks. The purpose of the survey was to examine consumers’ responses to information on the safety of raw milk cheeses. The associated consumer test provided information about specialty cheese consumers’ preferences and purchasing behavior. Half of the consumers tested were provided with cheese pairs that were identified as being made from unpasteurized and pasteurized milk. The other half evaluated samples that were identified only with random 3-digit codes. Overall, more consumers preferred the raw milk cheeses than the pasteurized milk cheeses. A larger portion of consumers indicated preferences for the raw milk cheese when the cheeses were labeled and thus they knew which samples were made from raw milk. Most of the consumers tested considered the raw milk cheeses to be less safe or did not know if raw milk cheeses were less safe. After being informed that the raw milk cheeses were produced by a process approved by the FDA (i.e., 60-d ripening), most consumers with concerns stated that they believed raw milk cheeses to be safe. When marketing cheese made from raw milk, producers should inform consumers that raw milk cheese is produced by an FDA-approved process.     

Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the C_t values and histamine concentrations determined by HPLC.     

Effect of pasteurization of Ewe's milk and use of a native starter culture on the volatile components and sensory characteristics of roncal cheese.

The effect of pasteurization of milk and use of a native starter culture on the volatile components and sensory characteristics of a Spanish ewe's-milk cheese were examined. Three cheese batches were made, one from raw milk, another from pasteurized milk, and a third from pasteurized milk with an added native starter culture in addition to the commercial starter. Cheeses were analyzed at 1, 120, and 240 d of ripening. Analysis of the volatile components was by purge and trap connected to a gas chromatograph with a mass spectrometer and disclosed a total of 76 components belonging to the following chemical families: hydrocarbons, fatty acids, esters, sulfur and carbonyl compounds, and, in particular, alcohols. Pasteurization lowered the levels of certain volatile components, especially alcohols, aldehydes, and ketones. The cheeses made from pasteurized milk showed lower scores for attributes of characteristic taste and aftertaste, as well as a characteristic aroma at 240 d of ripening. These results suggest that the components present in higher concentrations in the cheeses made from the raw milk were necessary for development of characteristic Roncal cheese aroma. The new native starter culture tested did not exert a significant effect on any of the parameters considered, with the exception of certain isolated components, for which higher or lower quantities were recorded in the cheeses made with that starter culture, although the differences did not have a definite effect on the sensory characteristics of the cheeses.     

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 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.     

Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening

Two commercial starters were investigated for their potential ability to decarboxylate amino acids during goat cheese ripening. Two batches of goat cheese were produced with identical pasteurized milk but different starter cultures. One of them contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and the other Lactococcus lactis subsp. lactis. The amine contents, microbial counts, proteolysis-related parameters, pH, total solids and salt content were studied in raw materials and cheeses. In raw materials, polyamines were the prevailing amines, whereas the main amines in cheeses were putrescine, tryptamine and, in particular, tyramine (94.59 mg/kg). Aerobic mesophilic microorganisms and Lactococcus counts increased throughout ripening, while Enterobacteriaceae were no longer detectable in cheese after 30 days of ripening. Amine concentration rose during cheese ripening in both batches. Moreover, the decarboxylase activity of microorganisms isolated from samples during cheese ripening was assayed and discussed.     

Evolution of free amino acid content during ripening of Mahon cheese

Evolution of free amino acids (FAA) during ripening (10–300 days) of five batches of Mahon cheese was studied; three batches were made from raw milk and two from pasteurized milk. Major FAA were GLU, VAL, LEU, LYS and PHE (43–67% of total FAA). Relative THR, SER, GLU and GLY contents significantly increased during ripening whereas LEU, PHE and ORN decreased. GLU, ILE, SER, THR, GLY and PHE varied during ripening according to a zero order reaction (r² > 0.99). Principal component analysis showed that the first principal component could be considered representative of the ripening time. The most ripened cheeses showed high GLU, GLY, SER and THR contents and the less ripened cheeses presented high ORN, LEU, PHE and ALA contents. The second component distinguished between cheeses made with pasteurized milk (with high ASN and GLN contents) and raw milk. Indigenous microflora of raw milk showed a strong influence on the proteolytic activity in Mahon cheese.     

Specific effect of high-pressure treatment of milk on cheese proteolysis

The extent of primary and secondary proteolysis of cheeses made from raw (RA), pasteurized (PA, 72 degrees C, 15 s) or pressure-treated (PR, 500 MPa, 15 min, 20 degrees C) goats' milk was assessed. Modifications in cheese-making technology were introduced to obtain cheeses with the same moisture content, and thus studied per se the effect of milk treatment on cheese proteolysis.The PR milk cheese samples were differentiated from RA and PA milk cheeses by their elevated beta-lg content, and by the faster degradation of alphas1-, alphas2- and beta-CN throughout ripening. Non-significant differences were found in either pH 4.6 soluble-nitrogen or trichloracetic acid soluble-nitrogen contents of cheeses. However, the pasteurization of milk decreased the free amino acid production in cheese. The RA milk cheeses had the highest amount of proline and the lowest concentrations of serine, tyrosine, arginine and alpha-aminobutyric acid, whereas PR milk cheese showed higher levels of arginine.     

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.     

Volatile fraction and sensory characteristics of Manchego cheese. 1. Comparison of raw and pasteurized milk cheese

Manchego cheese can be manufactured from raw or pasteurized ewes' milk. An automatic purge and trap apparatus, coupled to a GC-MS was used to isolate. identify and compare the relative amounts of the volatile components of raw and pasteurized Manchego cheese during ripening. The majority of volatile compounds were more abundant in raw milk (RM) cheeses than in pasteurized milk (PM) cheeses. Alcohols and esters predominated in the profile of RM Manchego cheese, while methyl-ketones and 2,3-butanedione were quantitatively important in PM cheeses. Branched chain alcohols were much more abundant in RM cheeses. The discriminant analysis separated 100% samples into RM or PM cheeses by using only 16 volatile compounds. Aroma intensity was correlated with esters, branched chain aldehydes and branched chain alcohols in RM cheeses, and with esters, branched chain aldehydes, 2-methyl ketones and 2-alkanols in PM cheeses. Diacetyl was positively correlated with the aroma attribute 'toasted' and negatively correlated with aroma quality in PM cheeses.     

The effects of starter culture on chemical composition, microbiological and sensory characteristics of Turkish Kaşar Cheese during ripening

Kaşar cheese samples were produced from raw milk and starter culture-added pasteurized milk. Chemical, microbiological and organoleptic properties of kaşar cheeses were analysed at certain times during the ripening periods (on the 1st, 7th, 15th, 30th, 60th, 90th days). Generally, chemical parameters were not affected by starter culture. The pH, ripening index, water-soluble nitrogen and non-protein nitrogen did not show significant differences between the cheese samples. The addition of starter affected the microbiological quality of the cheeses. Starter culture-added kaşar cheeses contained low levels of total aerobic mesophilic bacteria, moulds and yeasts, and coliforms, and achieved higher organoleptic scores than those of cheeses made from raw milk. The starter cultures contributed to acidity and microbial quality of the cheese.     

Effects of milk high pressure homogenization on biogenic amine accumulation during ripening of ovine and bovine Italian cheeses

The aim of this work was to evaluate the biogenic amine (BA) content during the ripening of both bovine and ovine cheeses obtained using milk subjected to a homogenization treatment at 100 MPa before cheese-making. The data obtained were compared with those from cheeses produced by the same milks without any treatment or thermized. The results showed that both microbial ecology and BA concentrations of cheeses during ripening were significantly influenced by the type of milk used for cheese-making and by the treatment applied to the raw materials. In particular, the microbial counts found in Caciotta indicated that the high pressure homogenization (HPH) of milk significantly reduced the presence of the yeasts, Micrococcaceae and lactobacilli at the end of ripening. On the other hand, the HPH treatment of milk favoured the proliferation of yeasts in ovine cheese. Moreover, the ovine cheeses were characterized by a remarkably higher accumulation of BA than bovine cheeses. However, the HPH treatment of milk was able to drastically reduce the biogenic amine concentrations in both cheese typologies at the end of ripening.     

Ripened Semihard Cheese Covered with Lard and Dehydrated Rosemary (Rosmarinus officinalis L.) Leaves: Processing,Characterization, and Quality Traits

This study aimed to establish a manufacturing protocol and to characterize semihard cheese covered with lard and rosemary during ripening. After the manufacturing protocol was defined, the cheeses were produced with pasteurized and raw milk from Holstein cows, with and without (control) coating, and then ripened for 60 d. During this period the physicochemical properties, color, proteolysis, texture profile, and sensory acceptance were performed. The early‐ripening cheeses differed from the others in terms of color and moisture content. Multivariate statistical analysis separated chesses in groups differentiated by the effects of heat treatment of milk and ripening period. The ripened cheeses obtained from raw milk were sensorially more preferred. The coating gave the final products higher moisture content and favored color and texture characteristics. Consumer testing showed that the cheese obtained from raw milk and coated with lard and rosemary was the most preferred (acceptance of 82%) due to the specific coating of rosemary (aroma and flavor). This product has potential to add value and to diversify the production of semihard cheeses.     

Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk.

The ability of Salmonella Enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10(4) CFU/ml of a luminescent strain of Salmonella Enteritidis (lux) and 10(8) PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella Enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella Enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella Enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella Enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella Enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella Enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10(3) CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk.     

牦牛乳硬质干酪成熟过程中生物胺与细菌群落结构分析

成熟干酪易产生生物胺,生物胺对人体健康具有影响,因此,为了探究牦牛乳硬质干酪的质量安全性,本实验利用高效液相色谱和高通量测序技术对干酪成熟过程中生物胺和细菌群落结构进行测定和分析。结果表明：牦牛乳硬质干酪中主要生物胺为腐胺、2-苯乙胺、酪胺、组胺和尸胺,在1～6 个月成熟过程中,干酪中各生物胺含量呈现增加趋势。干酪中生物胺含量最高阶段均出现在成熟期5～6 个月。干酪中组胺、酪胺和总生物胺含量分别低于推荐安全剂量50、100 mg/kg和1 000 mg/kg。干酪中游离氨基酸含量与除组胺之外的各生物胺含量、总生物胺含量、成熟时间之间具有显著正相关性（P＜0.01,P＜0.05）,各生物胺含量之间也存在正相关性。不同成熟期干酪包含相同属的微生物,其中链球菌属（Streptococcus）为优势菌属,平均相对丰度为84.63%,明串珠菌属（Leuconostoc）次之,平均相对丰度为6.91%,其他分别为乳杆菌属（Lactobacillus）、拉乌尔菌属（Raoultella）、不动杆菌属（Acinetobacter）、假单胞菌属（Pseudomonas）、肠杆菌属（Enterobacter）和无分类的肠杆菌科（unclassified_Eunnterobacteriaceae）。不同成熟期干酪中各菌属的相对丰度具有差异。本研究可为评估牦牛乳硬质干酪的质量安全性和生物胺形成机理提供理论依据。