Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

Comparison of growth and survival of single strains of Lactococcus lactis and Lactococcus cremoris during Cheddar cheese manufacture

Traditionally, starter cultures for Cheddar cheese are combinations of Lactococcus lactis and Lactococcus cremoris. Our goal was to compare growth and survival of individual strains during cheesemaking, and after salting and pressing. Cultures used were 2 strains of L. lactis (SSM 7605, SSM 7436) and 2 strains of L. cremoris (SSM 7136, SSM 7661). A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2, and 3.6% from which were selected cheeses with salt-in-moisture levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheesemaking and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than species dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ～4 times (～0.6 log) more bacterial cells than those made using L. cremoris strains. Growth of the strains used in this study was not influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ～5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 3 to 19% of the cells were designated as being live, but semipermeable, with L. cremoris strains having the higher number of semipermeable cells.     

乳酸乳球菌乳脂亚种MG1363中盐诱导启动子的克隆

为了从乳酸菌中筛选和克隆启动子,实验利用缺失T7启动子的质粒载体PRSET/LacZ直接在大肠杆菌（E.coli）DH5α中分离乳酸乳球菌乳脂亚种（Lactococcus lactis subsp.cremoris）MG1363的基因启动子片段,获得了10多个具有抗氨苄和盐诱导出蓝斑的重组子。反复筛选并对其中一个抗性最高的重组子PRSET-osm进行序列测定和同源性分析发现,所克隆的基因启动子片段来自乳酸乳球菌乳脂亚种MG1363的基因组,并具有原核启动子的保守序列（Pribnow框和Sextama框）。对启动子osm进行进一步序列分析和鉴定发现,其在大肠杆菌BL21中启动LacZ基因的表达,确定osm为盐诱导启动子。      

一株乳酸乳球菌所产细菌素的生物学特性

对乳酸乳球菌KLDS4.0326所产类细菌素进行分离纯化,并对细菌素的部分生物学特性进行研究。在排除酸性产物和过氧化氢的干扰后,菌株的无细胞发酵上清液对金黄色葡萄球菌和枯草芽孢杆菌等革兰氏阳性细菌以及大肠杆菌、沙门氏菌等革兰氏阴性细菌具有显著的抑制作用;经胰蛋白酶、胃蛋白酶、木瓜蛋白酶和蛋白酶K处理后,抑菌活性降低,表明抑菌成分为蛋白类物质;该细菌素具有良好的热稳定性,在酸性条件下抑菌活性稳定,并且具有较广的抑菌谱,能够抑制多种革兰氏阳性细菌和革兰氏阴性细菌。     

Ultrafiltered milk reduces bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide-producing culture

The objectives were to reduce bitterness in reduced-fat Cheddar cheese made with an exopolysaccharide (EPS)-producing culture and study relationships among ultra-filtration (UF), residual chymosin activity (RCA), and cheese bitterness. In previous studies, EPS-producing cultures improved the textural, melting, and viscoelastic properties of reduced-fat Cheddar cheese. However, the EPS-positive cheese developed bitterness after 2 to 3 mo of ripening due to increased RCA. We hypothesized that the reduced amount of chymosin needed to coagulate UF milk might result in reduced RCA and bitterness in cheese. Reduced-fat Cheddar cheeses were manufactured with EPS-producing and nonproducing cultures using skim milk or UF milk (1.2×) adjusted to a casein:fat ratio of 1.35. The EPS-producing culture increased moisture and RCA in reduced-fat Cheddar cheese. Lower RCA was found in cheese made from UF milk compared with that in cheese made from control milk. Ultrafiltration at a low concentration rate (1.2×) produced EPS-positive, reduced-fat cheese with similar RCA to that in the EPS-negative cheese. Slower proteolysis was observed in UF cheeses compared with non-UF cheeses. Panelists reported that UF EPS-positive cheese was less bitter than EPS-positive cheese made from control milk. This study showed that UF at a low concentration factor (1.2×) could successfully reduce bitterness in cheese containing a high moisture level. Because this technology reduced the RCA level (per g of protein) to a level similar to that in the control cheeses, the contribution of chymosin to cheese proteolysis would be similar in both cheeses.     

Intragenomic 16S rRNA gene heterogeneity in Lactococcus lactis subsp. cremoris

Partial 16S rRNA gene sequencing of Lactococcus lactis subsp. cremoris strains from our collection identified strains containing sequences diagnostic of both subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris together in the same strain. The presence of a plasmid-encoded 16S rRNA pseudogene partly explained this result. Twenty-four out of 46 L. lactis subsp. cremoris strains tested by PCR contained this pseudogene. However, further analysis showed that five of these 24 strains also contained chromosomal 16S rRNA genes with sequences typical of L. lactis subsp. lactis. Genetic and phenotypic tests indicated these strains were otherwise normal L. lactis subsp. cremoris strains. Past recombination events between the 16S rRNA pseudogene and chromosomal 16S rRNA genes may explain this phenomenon. Genomic heterogeneity for both 16S rRNA and other gene sequences was observed for L. lactis subsp. cremoris SK11 from different laboratories, indicating caution is needed when integrating data from diverse sources for nominally the same strain.     

Metabolism of Lactococcus lactis subsp. cremoris MG 1363 in acid stress conditions

The metabolism of glucose by Lactococcus lactis subsp. cremoris MG 1363 remains homolactic whatever the pH of the culture medium. The growth rate decreased with the acidification of the medium until a limit pH value of 4.0 for which no growth was observed. In contrast, the specific rate of glucose consumption decreased only for very low pH values, i.e., below 4.5. The efficiency of biomass synthesis relative to the energy supply decreased when the medium pH diminished, as illustrated by Y(ATP) values. This observation was related to the increase in both components of the proton-motive force when the pH decreased. The growth stopped when the internal pH reached a limit value of 5.4 due to organic acid accumulation.     

Effect of exopolysaccharides and proteolytic activity of Lactococcus lactis subsp. cremoris strains on the viscosity and structure of fermented milks

Two exopolysaccharide (EPS)-producing Lactococcus lactis subsp. cremoris strains (B35 and B891) were used to study the effect of the kinetics of EPS production and bacterial proteolytic activity on the structure of milk gels and the viscosity of stirred milk gels. Strains were grown at 20 °C in milk containing either yeast extract or casitone and at 30 °C in either milk alone or milk containing casitone. Lactococcal counts, pH decrease and production and molecular characteristics (molar mass and radius of gyration) of both EPSs were followed during milk fermentation. The level of proteolysis in the fermented milks was determined after 24 h of incubation. The results obtained showed that the yield of EPS and the timing of EPS production during milk-gel formation were the most important factors that influenced the structure of the milk gels and the viscosity of the stirred product. The proteolytic activity of the strains did not seem to play any significant role.     

The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis

The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1 °C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.     

Efficacy of four conjugal lactococcal phage resistance plasmids against phage in commercial Lactococcus lactis subsp. cremoris cheese starter strains

The efficacy of four lactococcal phage resistance plasmids (pNP40, pMU1311, pDI60 and pKP100) against phage was assessed after their conjugal transfer to four commercial Lactococcus lactis subsp. cremoris cheese starter strains and to the plasmid-free strain L. lactis subsp. cremoris MG1363. In MG1363, only pNP40 conferred resistance to prolate phages c2 and 643. Highest levels of resistance to small isometric phages in MG1363 occurred when pNP40 was stacked together with pMU1311 or pDI60. In the four starter strains, the plasmids conferred varying levels of resistance to small isometric phages. Growth and acidification rates in milk of most transconjugants derived from the starter strains decreased, but this was not always due to loss of plasmid-encoded cell wall proteinase (lactocepin) activity. Only one transconjugant grew during repeated subculture in milk with addition of factory wheys containing phages. This and the presence of bacteriocins encoded on pMU1311 and pDI60 limited application of the plasmids to protect L. lactis subsp. cremoris starters against phages in industry. However, some of the plasmids could be useful in extending the industry life of starters where fast acid production is not required or where bacteriocin production is acceptable.     

Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese

Cheddar-type cheeses were manufactured using fermentation-produced camel or calf chymosin. There were no significant differences in the composition and pH between the cheeses made with either coagulant. The extent of primary proteolysis was significantly lower in cheeses made with camel chymosin than in cheeses made with calf chymosin. There were large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar except for isoleucine, histidine and lysine. The cheeses made with camel chymosin were characterized by lower intensities of sulphur and brothy flavours and showed less bitter taste; however, the cheeses made with calf chymosin had greater breakdown of texture, higher smoothness and mouthcoating and were more cohesive and adhesive. The results of this study suggest that camel chymosin appears to be suitable for making Cheddar cheese with lower levels of proteolysis but with good flavour.     

Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening.To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed.We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively.The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.     

乳酸乳球菌乳脂亚种冷冻干燥保护剂优化及其贮藏稳定性

采用单因素试验、Plackett-Burman试验设计和Box-Behnken响应面法,筛选和优化乳酸乳球菌乳脂亚种KLDS4.0326真空冷冻干燥保护剂工艺,并分析冻干菌粉在不同温度下的贮藏稳定性。结果表明：脱脂乳粉、海藻糖和甘油含量是影响菌体细胞存活率的关键因素;以菌体细胞存活率为响应值,对3 个因素进行优化分析,保护剂最佳配比为：脱脂乳粉10.77 g/100 mL、海藻糖7.81 g/100 mL、甘油3.31 g/100 mL,此条件下菌体细胞存活率为（87.43±1.62）%,与理论预测值接近。在4 ℃和25 ℃条件下保藏12 个月后,菌体细胞存活率分别为40.63%和8.67%,冻干菌粉在4 ℃条件下具有更高贮藏稳定性。     

Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurised milk

Cheddar cheeses were made from raw (R1, R8) or pasteurised (P1, P8) milk and ripened at 1°C (P1, R1) or 8°C (P8, R8). Volatile compounds were extracted from 6 month-old cheeses and analysed, identified and quantified by gas chromatography-mass-spectrometry. A detailed sensory analysis of the cheeses was performed after 4 and 6 months of ripening. The R8 cheeses had the highest and P1 the lowest concentrations of most of the volatile compounds quantified (fatty acids, ketones, aldehydes, esters, alcohols, lactones and methional). The R8 and P8 cheeses contained higher levels of most of the volatiles than R1 and P1 cheeses. Ripening temperature and type of milk influenced most of the flavour and aroma attributes. Principal component analysis (PCA) of aroma and flavour attributes showed that P1 and R1 had similar aroma and flavour profiles, while R8 had the highest aroma and flavour intensities, highest acid aroma and sour flavour. The age of cheeses influenced the perception of creamy/milky and pungent aromas. PCA of the texture attributes separated cheeses on the basis of ripening temperature. The R8 and P8 cheeses received significantly higher scores for perceived maturity than P1 and R1 cheeses. The P1 and R1 cheeses had similar values for perceived maturity. In a related study, it was found that concentrations of amino acids and fatty acids were similar in R1 and P1 during most of the ripening period, and R1 and P1 cheeses had low numbers of non-starter lactic acid bacteria (NSLAB). The panel found that ripening temperature, type of milk and age of cheeses did not influence the acceptability of cheese. It is concluded that NSLAB contribute to the formation of volatile compounds and affect the aroma and flavour profiles and the perceived maturity of Cheddar cheese.     

Effect of various high-pressure treatments on the properties of reduced-fat Cheddar cheese

A major problem with reduced-fat cheese is the difficulty in attaining the characteristic flavor and texture of typical full-fat versions. Some previous studies have suggested that high hydrostatic pressure (HHP) can accelerate the ripening of full-fat cheeses. Our objective was to investigate the effect of HHP on reduced-fat (~7.3% fat) Cheddar cheese, with the goal of improving its flavor and texture. We used a central composite rotatable design with response surface methodology to study the effect of pressure and holding time on the rheological, physical, chemical, and microbial characteristics of reduced-fat Cheddar cheese. A 2-level factorial experimental design was chosen to study the effects of the independent variables (pressure and holding time). Pressures were varied from around 50 to 400 MPa and holding times ranged from 2.5 to 19.5 min. High pressure was applied 1 wk after cheese manufacture, and analyses were performed at 2 wk, and 1, 3, and 6 mo. The insoluble calcium content as a percentage of total Ca in cheeses were not affected by pressure treatment. Pressure applications ≥225 MPa resulted in softer cheese texture during ripening. Pressures ≥225 MPa increased melt, and resulted in higher maximum loss tangent values at 2 wk. Pressure treatment had a greater effect on cheese microbial and textural properties than holding time. High-pressure-treated cheeses also had higher pH values than the control. We did not observe any significant difference in rates of proteolysis between treatments. In conclusion, holding times of around 5 min and pressures of ≥225 MPa could potentially be used to improve the excessively firm texture of reduced-fat cheese.     

切达干酪加速成熟方法及其研究现状

概述了对切达干酪的加速成熟的现状与研究方法,通过提高温度、添加促熟酶、修饰发酵剂细胞、高压处理等方法缩短切达干酪的成熟时间,提高经济效益。     

快速成熟契达干酪成熟期间的理化特性变化

对天然契达(Cheddar)干酪快速成熟期间理化特性进行了研究,结果表明,在21 d内,pH值呈下降趋势(P<0.05),在21 d到35 d的成熟中,pH值缓慢增加(P>0.05);Cheddar干酪快速成熟过程中由于蛋白酶和脂肪酶的作用使蛋白发生水解,pH值为4.6醋酸溶液—水溶性氮质量分数、质量分数为12%的三氯乙酸—可溶性氮和游离氨基酸(FAA)的质量分数都呈明显增加趋势(P<0.05);成熟过程中TPA各项指标均有不同程度的变化,硬度和凝聚性呈先下降后增加的趋势,弹性和咀嚼性呈先上升后下降的趋势。