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。     

生物胺降解菌株的筛选与鉴定

从实验室保藏的不同来源乳酸菌菌株中筛选出具有生物胺降解能力的菌株,测定其在不同盐浓度、pH下的生长能力。结果表明,筛选出3株具有生物胺降解能力的菌株,经16SrDNA序列分析,菌株HM22鉴定为发酵乳杆菌(Lactobacillus fermentum),菌株HM24为植物乳杆菌(Lactobacillus plantarum),菌株YF10042为粪肠球菌(Enterococcus faecalis);其中植物乳杆菌HM24的降解能力最优,对色胺、苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺、精胺的降解率分别为(25.90±2.30)%,(35.75±2.71)%,(27.61±3.94)%,(25.91±3.76)%,(22.54±2.64)%,(34.55±1.90)%,(39.25±1.86)%,(55.66±7.08)%;当盐浓度为4%～10%时,3株菌株耐受力随盐浓度的增加而降低,;当pH为3.5～9.5时,3株菌株均可生长。     

冷藏乳制作的牦牛乳硬质干酪成熟过程中生物胺含量的变化

目的:揭示冷藏原料乳制作的干酪成熟过程中生物胺含量变化规律,评价其质量安全性。方法:以冷藏24,48,72 h牦牛乳制作的硬质干酪为研究对象,利用高效液相色谱法对干酪成熟过程中生物胺含量进行测定。结果:在0～6个月成熟过程中,不同冷藏牦牛乳制作的硬质干酪中生物胺含量呈升高趋势。牦牛乳冷藏时间从24 h延长到72 h时,其干酪中总生物胺、2-苯乙胺、尸胺、酪胺和腐胺含量也依次增大。成熟4个月后,冷藏72 h牦牛乳制作的硬质干酪中各生物胺含量明显高于其余两组干酪。成熟6个月时,冷藏72 h原料乳制作的牦牛乳硬质干酪中生物胺总量和酪胺含量分别为(212.94±8.03),(81.04±3.92) mg/kg。结论:随着原料乳冷藏时间和干酪成熟时间的延长,干酪中生物胺含量增多,但是原料乳冷藏时间低于72 h时,其干酪中生物胺含量低于学者建议含量。     

不同啤酒生物胺含量的比较

使用反相高效液相色谱法,测定啤酒中8种常见生物胺含量。以苯甲酰氯为衍生试剂,Agilent EclipseXDB-C18为固定相,乙腈和乙酸铵溶液为流动相,紫外检测波长设置为254 nm,梯度洗脱。本文首先对国内市售啤酒生物胺种类及含量范围进行研究,检测结果表明国内啤酒中8种生物胺均有存在,但不同样品所含种类不同,且有个别胺含量偏低无法定量。不同厂家的产品在种类和含量上存在差别,但整体含量接近,生物胺总量为4.21～10.59 mg/L,不足以对人类健康产生危害。原麦汁浓度、酒精度与生物胺含量的关系显示,二者与生物胺有一定联系,但非主要因素,相对于生产厂家和发酵情况,原麦汁浓度、酒精度对生物胺含量的影响较小。     

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.     

溶菌酶处理发酵剂加速干酪成熟的研究

以pH、蛋白质降解、脂肪酸含量变化及干酪的感官特征为指标,研究了溶菌酶处理发酵剂对干酪成熟的影响。结果表明:实验组干酪的pH在整个成熟期处于对照组1(添加2%发酵剂)干酪和对照组2(添加2.5%发酵剂)的pH之间,表明通过溶菌酶处理发酵剂可明显抑制干酪成熟期间的产酸量;添加溶菌酶处理发酵剂可加速干酪中蛋白质分解和乳脂肪的降解;感官评价结果表明,添加溶菌酶处理发酵剂可提高干酪风味,在相同的成熟时间实验组干酪的质地也较对照组好。     

干酪生产用发酵剂的筛选

概述了干酪的营养特点及生产用菌种所具备的特性,对本实验室现有的4种单一菌株和7种混合菌株的发酵性能和生化特性进行了研究,优选出了适于干酪加工的2株优良菌种L3（乳酸乳球菌）和L6（干酪乳杆菌：瑞士乳杆菌=1：1）。     

Formation of biogenic amines by proteolytic enterococci during cheese ripening

Six strains of Enterococcus faecalis and three strains of E faecium were investigated for their proteolytic activity and ability to decarboxylate the amino acids lysine, histidine, tyrosine, ornithine and phenylalanine. All strains tested displayed cell wall‐associated proteolytic activities, and three E faecalis strains displayed additionally extracellular proteolysis. None of the strains tested was able to form putrescine, histamine or cadaverine. Eight strains decarboxylated tyrosine, and two strains of E faecalis and one strain of E faecium formed phenylethylamine. Gouda cheese was produced from milk supplemented with two strains of E faecalis (10⁷ cfu ml⁻¹). Both strains were proteolytic and potent tyramine formers. Final viable counts of 10⁹ cfu g⁻¹ and tyramine concentrations of 477 mg kg⁻¹ were detected after a 12 week ripening period. © 1999 Society of Chemical Industry     

Casein breakdown during ripening of Idiazabal cheese: influence of starter and rennet type

The objective of this work was to determine the effect of starter and rennet type on casein breakdown during Idiazabal cheese ripening. Four batches of cheeses were manufactured with two rennets, commercial calf rennet and artisanal lamb rennet, and the use of natural flora or a commercial starter. Electrophoretic analysis of cheese samples showed six bands identified as α_s1‐, α_s2 + β‐, α_s1‐I‐, γ₁‐, β‐I‐ and para‐κ‐casein. As expected, the casein breakdown during cheese ripening was considerably affected by rennet type and the use of a commercial starter. The artisanal lamb rennet produced a higher hydrolysis of casein fractions than the commercial calf rennet, probably owing to its high percentage of chymosin (around 78%). The effect of addition of starter on proteolysis was dependent on the casein fractions generated by artisanal lamb rennet or commercial calf rennet. © 2000 Society of Chemical Industry     

Monitoring the chemical and textural changes during ripening of Iranian White cheese made with different concentrations of starter

The effect of the concentration of starter inoculated to milk on the composition, free tyrosine-tryptophan content, microstructure, opacity, and fracture stress of Iranian White cheese (IWC) was studied during 50 d of ripening in brine. Three treatments of cheese were made using 1-fold (IWC1S), 2-fold (IWC2S), and 4-fold (IWC4S) concentrations of a direct-to-vat mesophilic mixed culture containing Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis as starter. As ripening progressed, moisture and protein contents of the treatments continuously decreased, whereas their total ash, salt, and salt in moisture contents increased. Fat content and pH of cheeses remained stable during ripening. The pH of cheese milk at the time of renneting, which decreased by increasing the concentration of starter (6.57, 6.49, and 6.29 for IWC1S, IWC2S, and IWC4S, respectively), significantly affected most of the chemical characteristics and opacity of cheese. Lower pH values at renneting decreased moisture and ash contents, whereas cheese protein content increased. The concentration of free tyrosine-tryptophan in curd increased at first 29 d but decreased between d 29 and 49 of aging. The changes observed in cheese whiteness followed the changes in moisture content of the treatments. As the concentration of starter inoculated to milk increased, the value of fracture stress at a given ripening time significantly decreased, leading to a less resistant body against applied stress. A similar trend was also observed for fracture strain during cheese ripening. The micrographs taken by scanning electron microscopy provided a meaningful explanation for decrease in the value of fracture stress. As the cheese ripening progressed or the concentration of starter increased, the surface area occupied by the protein fraction in cheese microstructure decreased, leading the way to lower the force-bearing component in cheese texture.     

Effect of the lactoperoxidase system on the activity of mesophilic cheese starter cultures in goat milk

The effect of goats’ milk lactoperoxidase (LP) system on the activity of commercially available mesophilic cheese starter cultures was investigated. The growth and acid production of the starter cultures were measured at 2 h intervals for 8 h in goats’ milk kept at 30°C. Most of the starter cultures examined were found to be sensitive to the LP system, but varied in their susceptibility to inhibition. The activity of the mixed starter cultures CHN11, CHN22, CHN19, DCC240 and Flora Danica Normal was strongly inhibited by the LP system. However, the mixed starter culture LL 50C showed resistance to the LP system. The single strain culture Lactococcus lactis subsp. lactis NCDO 605 was inhibited by the LP system. However, the cultures Lactococcus lactis subsp. diacetylactis NCDO 176 and Leuconostoc mesenteroides subsp. cremoris ATCC 33313 were insensitive to the LP system. The results of this study indicate the need for routine screening of starter cultures for resistance to the LP system before using them for cheesemaking from goats’ milk preserved by the LP system.     

超高效液相色谱-串联质谱法同时测定蜂蜜中8种生物胺

建立了同时测定蜂蜜中色胺(TR)、2-苯乙胺(PHE)、腐胺(PUT)、尸胺(CAD)、组胺(HIS)、酪胺(TYR)、亚精胺(SPD)和精胺(SP)8种生物胺的超高效液相色谱串联质谱柱前衍生的分析方法。样品经5%三氯乙酸溶液提取后用丹磺酰氯柱前衍生,采用Agilent Poroshell 120 SB-C₁₈(50 mm×2.1 mm,2.7 μm)柱,以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相进行定性和定量分析。采用本方法对22种市售蜂蜜进行检测,结果显示8种生物胺在不同的蜂蜜中检出,所有蜂蜜样品中均检测到2-苯乙胺和腐胺,其中2-苯乙胺也是蜂蜜样品中平均含量最多的生物胺。本方法中8种生物胺的线性相关系数均大于0.99,检出限为0.01~0.51 μg/L。在添加水平为100、500、1000 μg/kg时,样品的平均回收率在76%~112%之间,日内精密度小于等于9.8%,日间精密度小于等于13.1%。该方法快速简单,灵敏准确,且回收率稳定,适用于蜂蜜中生物胺的检测。     

超高效液相色谱法测定不同品种桃果酒中8种生物胺含量

摘要：目的 建立一种检测桃果酒中生物胺的超高效液相色谱分析方法, 并对不同品种桃果酒的生物胺组成与含量进行测定。方法 样品采用丹磺酰氯衍生, 乙酸乙酯液-液萃取, Waters ACQUITY UPLC BEH C18色谱柱(2.1 mm×100 mm, 1.7 μm)分离, 柱温35℃, 乙腈-0.1%乙酸水溶液作流动相, 流速0.2 mL/min, 梯度洗脱, 紫外检测波长254 nm,外标法定量。 结果 柱前丹磺酰氯衍生超高效液相色谱-紫外法可在16 min内完成色胺、β-苯乙胺、腐胺、尸胺、组胺、酪胺、亚精胺、精胺等8种生物胺的测定, 检出限在15.76~21.43μg/L之间, 定量限在53.58~71.07μg/L之间; 日内精密度在0.170.85%~1.883.31%之间, 日间精密度在0.601.02%~2.824.36%之间; 加标回收率在87.8389.71%~108.22104.85%之间, 相对标准偏差RSD≤3.36%。10个品种桃果酒中均可检测到8种生物胺, 其中以色胺、腐胺含量较高, 而酪胺、精胺含量较低。白凤、武汉2号桃果酒中生物胺总胺含量较高, 而春蜜、中桃绯玉桃果酒中总胺含量较低。结论 该方法快速简便、灵敏高效、准确度高、重现性好, 可用于桃果酒中生物胺的分析检测。不同品种桃果酒的生物胺组成与含量差异较大, 选择适宜的原料品种有助于控制桃果酒中生物胺含量。     

Effect of forage type,season, and ripening time on selected quality properties of sheep milk cheese

The aim of this research was to study changes in the microbial populations, free AA profile, biogenic amine content, and sensory characteristics of ripened cheeses (100 and 180 d) produced in different seasons (summer, autumn, winter, and spring) from pasteurized sheep milk from 8 commercial flocks fed hay or silage diets. Twenty-one individual AA and 6 biogenic amines were determined by ultra-high performance liquid chromatography. Type of conserved forage for sheep feeding did not affect the variables studied, which is of great interest because hay and silage are low-cost ingredients for sheep feeding. Proteolysis led total free AA concentrations ranging between 35,179.26 and 138,063.71 mg/kg of cheese at 180 d of ripening. γ-Aminobutyric acid, which has been associated with beneficial effects on human health, was the second most abundant AA in all cheese samples, accounting for 15% of total free AA. Spring cheeses showed 2-fold higher concentrations of γ-aminobutyric acid than summer and autumn cheeses at the end of ripening. Overall, spring, winter, and autumn cheeses had lower average concentration of biogenic amines (431.99 mg/kg of cheese) than summer cheeses (825.70 mg/kg of cheese) as well as better sensory characteristics. Therefore, this study could provide the dairy industry with useful information for producing cheeses with valuable nutritional and sensory quality for consumers.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.     

大蒜精油和发酵剂对熏马肠中生物胺及微生物分布的影响

为研究大蒜精油和发酵剂对熏马肠中生物胺的影响,采用高效液相色谱法测定熏马肠中生物胺含量,利用 宏基因组学技术分析熏马肠中微生物多样性和生物胺含量的变化情况。结果表明：将大蒜精油和发酵剂联合使用能 够很好地减少熏马肠中尸胺、腐胺、组胺、酪胺和苯乙胺的产生,对控制生物胺在熏马肠中的含量起到非常显著的 作用;熏马肠中含有丰富的微生物物种,通过样品科分类水平物种分析,得出乳酸菌在熏马肠整个加工过程都是优 势菌种,而葡萄球菌在成熟后期数量较多。     

Physicochemical and textural characteristics and volatile compounds of semihard goat cheese as affected by starter cultures

Today, cheese is valued because of its high nutritional value and unique characteristics. Improving the texture and flavor of cheese by selecting suitable starter cultures is an important way to promote the development of cheese industry. The effect of starter cultures on the physicochemical and textural properties and volatile compounds during the ripening of semihard goat cheese were investigated in this work. Different starter cultures—mesophilic (M) and thermophilic starters (T), Lactobacillus plantarum ssp. plantarum ATCC 14917 (Lp), a mix of the M and T starters (M1), and mix of the M, T, and Lp starters (M2)—were used in the production of the goat cheeses. Volatile compounds were determined by a solid-phase microextraction/gas chromatography-mass spectrometric (SPME/GC-MS) method. The results showed that the moisture content of cheeses produced with the 5 kinds of starter cultures decreased after maturation, whereas ash content increased. The pH values of goat cheeses decreased first and then increased during maturity, and the pH value of M2 cheese was the lowest among the cheeses. The hardness and chewiness of the cheeses increased with increasing maturity, whereas cohesiveness, springiness, and resilience showed the opposite tendency. The 60-d-old cheese made with Lp had the highest chewiness, cohesiveness, springiness, and resilience, whereas the 60-d-old cheese made with M2 had the highest hardness. A total of 53 volatile components were identified by SPME/GC-MS, and carboxylic acids, alcohols, ketones, and esters were the 4 major contributors to the characteristic flavors of the cheeses. Volatile components and their contents differed greatly among the produced cheeses. The M2 cheese contained the highest relative content of the main volatile compounds (90.10%), especially butanoic acid and acetoin. Through a comprehensive comparison of the results, we concluded that M2 cheese had a dense texture and milky flavor, and M2 is a potential starter culture candidate for the production of goat cheese.     

新型脂肪胺荧光标记试剂的合成及小分子 脂肪胺的衍生检测

目的合成一种新的以荧光素为荧光团,N-羟基琥珀酰亚胺活性酯为反应基团的脂肪胺荧光标记试剂,并建立以新试剂标记短链脂肪胺并用高效液相色谱-荧光检测法进行分离的方法。方法在p H 8.5的Na2B4O7-H3BO3缓冲溶液中,25℃下,衍生试剂浓度为50μmol/L,衍生反应进行10 min,衍生物峰面积即可达到最大且稳定。结果在优化的条件下,建立的检测方法的线性范围为0.01~2μmol/L,线性回归系数0.999,检测限达到0.1~1μg/L。结论衍生后的产物利用高效液相色谱-荧光检测法,可以使得6种小分子脂肪胺得到有效分离,方法具有衍生速度快,灵敏度高等优点。