Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture

Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.     

发酵剂对牦牛乳硬质干酪成熟过程中生物胺的影响

乳酸菌产生物胺的能力具有菌株特异性,因此,为了探究不同种类发酵剂对牦牛乳硬质干酪中生物胺形成的影响,该试验利用高效液相色谱对3种不同发酵剂制作的硬质干酪成熟过程中生物胺进行了测定和分析。结果表明,嗜热和嗜温发酵剂牦牛乳硬质干酪中检测出2-苯乙胺、腐胺、尸胺、组胺和酪胺,混合发酵剂干酪中检测出腐胺、2-苯乙胺、尸胺和酪胺。各生物胺之间呈现正相关性。3种不同发酵剂干酪在1～6个月成熟过程中,其各生物胺整体呈现增加趋势,嗜热、嗜温和混合发酵剂干酪中总生物胺最高含量分别为(448.3±9.6)、(456.8±58.4)、(293±24.5)mg/kg。组胺和酪胺是2种毒性相对高的生物胺,嗜热发酵剂干酪中组胺和嗜温发酵剂干酪中酪胺最高,其最高含量分别为(20.8±7.9)、(92.9±6.7)mg/kg,混合发酵干酪中未检测出组胺,酪胺含量次之,3种不同发酵剂干酪中组胺、酪胺含量均低于推荐安全剂量50 mg/kg和100 mg/kg。这为合理选择发酵剂和控制干酪中生物胺形成提供了依据。     

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).     

Detection of biogenic amine producer bacteria in a typical Italian goat cheese

The aim of this study was to research decarboxylating bacterial strains and biogenic amine content in a typical Italian goat cheese (Robiola di Roccaverano). The study was performed on fresh and ripened samples of goat cheese manufactured from industrial and artisanal producers. Sixty-seven bacterial strains isolated showed decarboxylating activity, and Enterococcus faecalis was the most widespread decarboxylating species in all artisanal and industrial products. Pediococcus acidilactici and Enterococcus malodoratus were also identified as biogenic amine producers in Robiola di Roccaverano cheese. All the E. faecalis strains isolated in this study were able to decarboxylate tyrosine. Tyramine was the most abundant biogenic amine in cheese samples, while histamine was the most widespread. High amounts of these two biogenic amines were found in ripened samples (up to 2,067 mg/kg for tyramine and 1,786 mg/kg for histamine), whereas 2-phenylethylamine and tryptamine were present in almost all ripened cheeses at low concentrations. The detection of strains producing biogenic amines and the high concentrations of tyramine and histamine found in ripened Robiola di Roccaverano could represent a potential risk to the consumer.     

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.     

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.     

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。     

不同NaCl添加量对牦牛乳硬质干酪成熟过程中生物胺产生的影响

以不同盐添加量（1.0%、1.3%、1.8%和2.3%）牦牛乳硬质干酪为研究对象,分析牦牛乳硬质干酪0～6 个月成熟过程中生物胺的动态变化,并对干酪中产胺微生物进行筛选。结果显示：不同盐添加量牦牛乳硬质干酪中生物胺主要为色胺、β-苯乙胺、尸胺、酪胺和腐胺,未检测到组胺,生物胺积累阶段主要集中在成熟后期。不同盐添加量干酪中色胺含量最低,且在成熟后期含量差异较小。当盐添加量从2.3%减少到1.0%时,干酪中β-苯乙胺含量减少。盐添加量分别为1.0%和1.3%时,干酪中尸胺含量较低,且未检测出腐胺。当盐添加量在1.8%～2.3%时,随着盐添加量的增加,干酪中尸胺和腐胺含量整体呈现增加趋势。不同盐添加量干酪成熟过程中,其酪胺含量范围为3.13～49.81 mg/kg,且盐添加量为1.3%干酪中酪胺含量较高。不同盐添加量干酪中生物胺总量最高为304.18 mg/kg。采用显色培养基筛选出一株产胺微生物,经分子生物学鉴定为Enterococcus durans。     

Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic

The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25 mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewe’s milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200 mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumer’s health.     

Effect of Using Frozen Concentrated Direct-to-the-Vat Culture on the Yield and Quality of Cheddar Cheese

Approximately 53,000 liters (14,000 gal) pasteurized whole milk was used to make 16 batches of cheese, eight control batches using conventional bulk starter and eight batches using a frozen concentrated direct-to-the-vat culture. The calculated yields were based on both the weight of milk and cheese, and on the weight of milk solids and cheese solids. The yields averaged, respectively, 9.517% and 48.608% with the bulk starter and 9.707% and 49.191% with the frozen concentrated culture. These differences, slightly favoring the frozen concentrated direct-to-the-vat culture, can be of economic and commercial significance. The compositions of the cheeses made with the two starter cultures were very similar. Sensory evaluation did not reveal significant differences in flavor and texture of the two types of cheese when examined at 3, 6, 9 and 12 months.     

Content and distribution of biogenic amines in Dutch-type hard cheese

Content of biogenic amines (BA; histamine, tyramine, tryptamine, putrescine, 2-phenylethylamine, cadaverine, spermidine, spermine) and counts of microorganisms (total aerobic and facultative anaerobic mesophilic microorganisms, TAC; lactic acid bacteria, LAB; coliforms; total anaerobic microorganisms, TAN; yeasts + moulds), were evaluated within the ripening interval of 1–6 months in Dutch-type hard cheese, produced by two different producers (the cheeses designated as R30 and R45) using pasteurized milk and different starter cultures (designated as R30-FD and R30-CH in the case of the R30 cheeses). The cheeses from the two producers differed in their fat content (30% and 45% of fat in dry matter, respectively).     

Profile of Biogenic Amines in Goat Cheese Made from Pasteurized and Pressurized Milks

ABSTRACT: The formation of biogenic amines in goat cheese can be prevented through ensuring hygiene in raw materials and during manufacture, thereby avoiding potential decarboxylating microorganisms. High-pressure treatment may be used to inactivate microorganisms and is a potential alternative to pasteurization. This treatment could provoke higher proteolysis than pasteurization, leading to a higher availability of biogenic amine precursors. We compared the biogenic amine profile throughout the ripening of goat cheese made from pressurized milk with that obtained from pasteurized milk. Results indicate that the profile of biogenic amines is very similar for both cheeses. Tyramine was the prevailing amine in both cheeses, followed by cadaverine, putrescine, and histamine.     

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.     

Analysis of cheese for histamine, tyramine, tryptamine, histidine, tyrosine, and tryptophane

A method is described for determining the content of selected biologically active amines (histamine, tyramine, tryptamine) and amino acids (histidine, tyrosine, tryptophane) in cheeses by high performance liquid chromatography. The amines and amino acids were quantified by employing a counter ion-containing mobile phase and by comparing peak areas of high performance liquid chromatography charts for sample cheeses versus standard cheeses containing known amounts of added amines based on dual injections of samples and standards. Recovery of amines and amino acids varied from 87.5 to 111%. Histamine, which has been associated with food poisoning in concentrations of 185 mg/100 g in Swiss cheese and 180 to 500 mg/100 g in fish, was found in concentrations above 500 mg/100 g in Swiss cheese. The high performance liquid chromatography analytical method should be useful for screening to detect cheese samples containing toxic amounts of histamine and for research studies designed to determine the cause and effect relationships for histamine production in cheese.     

Biogenic amines in Brazilian cheeses

The levels of biogenic amines in Brazilian cheeses were investigated for the first time. Ninety-two samples of 10 types of cheese were purchased at retail stores. The amines were extracted using hydrochloric acid, partitioned into diethyl ether, separated through reversed-phase ion-pair chromatography and quantified fluorimetrically after postcolumn derivatization with o-phthaldehyde. Overall, spermine was the most prevalent amine (93%), followed by histamine (65%), 2-phenylethylamine (62%), spermidine (61%), putrescine (60%), cadaverine (59%), serotonin (44%), agmatine (38%), tyramine (37%) and tryptamine (29%). Spermine, agmatine, 2-phenylethylamine, serotonin, spermidine and tryptamine were detected at low levels (< 4.10 mg/100g). Cadaverine, tyramine, histamine and putrescine were present at levels up to 111.00, 21.25, 19.65 and 17.37 mg/100g, respectively. There was variability on the type and levels of amine in each kind of cheese suggesting that amine formation and accumulation in cheese could be prevented. Efforts should be made to understand amine formation in cheese in order to optimize technology and secure low amine levels. Susceptible individuals should be advised to consume cheeses with low biogenic amines contents.     

处理发酵剂对促熟干酪中生物胺和游离氨基酸的影响

以0、30、60、90 d促熟干酪中生物胺（组胺、色胺、苯乙胺、尸胺、酪胺）和游离氨基酸含量为指标,研究了添加复合处理发酵剂对干酪产生游离氨基酸和生物胺量的影响。结果表明：不同处理发酵剂添加量对各种生物胺产生的影响不同;添加处理发酵剂干酪中生物胺和游离氨基酸含量增加,其含量随着复合处理发酵剂添加量增加而增加。     

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.     

Determination of Biogenic Amines in Herby Cheese

Herby cheese (Otlu peynir) is widely produced and consumed in eastern parts of Turkey, and is generally made from sheep milk. The biogenic amines and organic acids content, microbiological, and chemical properties of Herby cheese were determined on 30 samples obtained from retail markets in East Anatolia Region, Turkey. The most important biogenic amines were tyramine (range 18.0–1125.5 mg kg^?1), followed by cadaverine varied from not detected to 1844.5 mg kg^?1. Histamine content generally was found higher than 100 mg kg^?1. The concentration of amines in some cheeses was much higher than the toxic dose limits. Generally, total amine content was found high in samples that had high nitrogen fraction and organic acids value. Levels of biogenic amines were significantly correlated with the organic acids of Herby cheese. The basic organic acid was found the lactic acid ranging from 585.4 to 26480.3 mg kg^?1, followed by butyric acid varying between 314.8 and 7329.6 mg kg^?1 values. Lipolysis and nitrogen fraction were determined high in ripened Herby cheese samples. Coliform counts of samples showed lower than 100 cfu g^?1 in the samples. Significant (p < 0.01) positive correlation was observed between acetic acid and phenylethylamine, putrescine, cadaverin, histamine, and tyramine.     

Influence of salt-to-moisture ratio on starter culture and calcium lactate crystal formation

The occurrence of l(+)-lactate crystals in hard cheeses continues to be an expense to the cheese industry. Salt tolerance of the starter culture and the salt-to-moisture ratio (S:M) in cheese dictate the final pH of cheese, which influences calcium lactate crystal (CLC) formation. This research investigates these interactions on the occurrence of CLC. A commercial starter was selected based on its sensitivity to salt, less than and greater than 4.0% S:M. Cheddar cheese was made by using either whole milk (3.25% protein, 3.85% fat) or whole milk supplemented with cream and ultrafiltered milk (4.50% protein, 5.30% fat). Calculated amounts of salt were added at milling (pH 5.40 ± 0.02) to obtain cheeses with less than 3.6% and greater than 4.5% S:M. Total and soluble calcium, total lactic acid, and pH were measured and the development of CLC was monitored in cheeses. All cheeses were vacuum packaged and gas flushed with nitrogen gas and aged at 7.2°C for 15 wk. Concentration of total lactic acid in high S:M cheeses ranged from 0.73 to 0.80 g/100 g of cheese, whereas that in low S:M cheeses ranged from 1.86 to 1.97 g/100 g of cheese at the end of 15 wk of aging because of the salt sensitivity of the starter culture. Concentrated milk cheeses with low and high S:M exhibited a 30 to 28% increase in total calcium (1,242 and 1,239 mg/100 g of cheese, respectively) compared with whole milk cheeses with low and high S:M (954 and 967 mg/100 g of cheese, respectively) throughout aging. Soluble calcium was 41 to 35% greater in low S:M cheeses (low-salt whole milk cheese and low-salt concentrated milk cheese; 496 and 524 mg/100 g of cheese, respectively) compared with high S:M cheeses (high-salt whole milk cheese and high-salt concentrated milk cheese; 351 and 387 mg/100 g of cheese, respectively). Because of the lower pH of the low S:M cheeses, CLC were observed in low S:M cheeses. However, the greatest intensity of CLC was observed in gas-flushed cheeses made with milk containing increased protein concentration because of the increased content of calcium available for CLC formation. These results show that the occurrence of CLC is dependent on cheese milk concentration and pH of the cheese, which can be influenced by S:M and cheese microflora.     

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.