A model to assess lactic acid bacteria aminopeptidase activities in Parmigiano Reggiano cheese during ripening

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.     

Nonstarter lactic acid bacteria and aging temperature affect calcium lactate crystallization in cheddar cheese

The occurrence of unappetizing calcium lactate crystals in Cheddar cheese is a challenge and expense to manufacturers, and this research was designed to understand their origin. It was hypothesized that nonstarter lactic acid bacteria (NSLAB) affect calcium lactate crystallization (CLC) by producing D(-)-lactate. This study was designed to understand the effect of NSLAB growth and aging temperature on CLC. Cheeses were made from milk inoculated with Lactococcus lactis starter culture, with or without Lactobacillus curvatus or L. helveticus WSU19 adjunct cultures. Cheeses were aged at 4 or 13 degrees C for 28 d, then half of the cheeses from 4 and 13 degrees C were transferred to 13 and 4 degrees C, respectively, for the remainder of aging. The form of lactate in cheeses without adjunct culture or with L. helveticus WSU19 was predominantly L(+)-lactate (> 95%, wt/wt), and crystals were not observed within 70 d. While initial lactate in cheeses containingL. curvatus was only L(+)-lactate, the concentration of D(-)-lactate increased during aging. After 28 d, a racemic mixture of D/L-lactate was measured in cheeses containing L. curvatus; at the same time, CLC was observed. The earliest and most extensive CLC occurred on cheeses aged at 13 degrees C for 28 d then transferred to 4 degrees C. These results showed that production of D(-)-lactate by NSLAB, and aging temperature affect CLC in maturing Cheddar cheese.     

干酪中非发酵剂乳酸菌

非发酵剂乳酸菌是干酪中主要的次生菌群，不属于发酵微生物，通常不能很好的在牛奶中生长，不能产酸，但对干酪的风味形成有很重要作用。本文概述了非发酵剂乳酸菌的一些特征，包括不同原料制成的干酪中非发酵剂乳酸菌的来源、生长能源的利用、自身存在的蛋白分解系统对干酪风味形成影响及作为一种提高干酪品质的附属发酵剂的应用展望。     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

用于低脂干酪开发的附属发酵剂的筛选

目的筛选出生产低脂干酪所需添加较为适宜的附属发酵剂。方法选择7株乳酸菌作为研究对象,测定其总肽酶活力、菌株生产能力、产酸能力、产粘能力、菌体自溶度、产胞外多糖数量、蛋白质水解能力等7种指标。结果 7株乳酸菌中嗜酸乳杆菌(Lactobacillus acidophilus,LA)总肽酶活力最高,为(256.46±11.88)μg/L,植物乳杆菌(Lactobacillus plantarum,LP)最低;菌株生长能力LA最好;菌株副干酪乳杆菌(Lactobacillus paracasei,LPC)在发酵10 h后的产酸能力最强且趋于稳定,pH在3.8左右;LP菌株产粘能力最优,为(168.10±14.72)mPa·s;瑞士乳杆菌(Lactobacillus helveticus,LH)菌株自溶度最好,达到35%;产胞外多糖数量最优菌株是LP,含量为(792.69±35.94)mg/L;LH的蛋白质水解能力最高,水解产生的游离氨基酸含量为(94.78±2.82)mg/L。结论 LH作为低脂干酪的附属发酵剂较好。     

青稞酒酒曲与奶酪中乳酸菌的筛选与应用

通过传统的分离、培养方法从西藏青稞酒酒曲和西藏、新疆奶酪中分别得到1株和3株乳酸菌,采用16S rRNA基因序列同源性分析的方法对菌株进行鉴定.研究进行了这4株乳酸菌发酵制备酸奶的比较实验,发现这4株乳酸菌均具备乳酸发酵制成酸奶的能力,而且制成的酸奶品质良好.表明从酒曲和奶酪中分离乳酸菌是筛选生产酸奶菌株的良好途径.丰富乳酸菌多样性,提供优良菌株,为酸奶行业的可持续发展提供保证.     

非发酵剂乳酸菌A-3和D-3对半硬质山羊奶干酪成熟的影响

目的获得加速半硬质山羊奶干酪成熟的非发酵剂乳酸菌菌株(non-starterlacticacidbacteria,NSLAB)。方法以前期分离自地中海地区山羊奶干酪中的2株优良NSLAB菌株为研究对象,测定其对干酪成熟过程中组成成分、微生物菌群、蛋白质水解和质构的影响。结果添加NSLAB菌株对干酪组成成分没有显著影响, NSLAB菌株没有影响乳球菌生长,在干酪成熟期间pH 4.6-SN和12%TCA-SN逐渐增加,且添加NSLAB的干酪在成熟30 d后显著增加了pH 4.6-SN和12%TCA-SN含量, 5%PTASN/TN的增加主要是由于乳酸菌中肽酶作用的结果, SDS-PAGE电泳结果说明添加NSLAB菌株的干酪中小分子多肽含量明显比对照干酪多,RP-HPLC分析得出干酪水溶性中肽的数量随着成熟时间增加。添加NSLAB菌株A-3没有改变干酪的硬度,使干酪的弹性增加。结论添加菌株A-3作为NSLAB的干酪样品中微生物自溶率高,蛋白水解程度强,质构性能良好,具有加速干酪成熟的潜力,是山羊奶干酪工业化生产的优良NSLAB。     

国外优质干酪中乳酸菌的分离鉴定与应用

对进口干酪中乳酸菌进行初步分离鉴定。同时利用其中优良菌株进行风味干酪生产小试，结果较好。     

Application of infrared microspectroscopy and multivariate analysis for monitoring the effect of adjunct cultures during Swiss cheese ripening

Improved cheese flavor has been attributed to the addition of adjunct cultures, which provide certain key enzymes for proteolysis and affect the dynamics of starter and nonstarter cultures. Infrared microspectroscopy provides unique fingerprint-like spectra for cheese samples and allows for rapid monitoring of cheese composition during ripening. The objective was to use infrared microspectroscopy and multivariate analysis to evaluate the effect of adjunct cultures on Swiss cheeses during ripening. Swiss cheeses, manufactured using a commercial starter culture combination and 1 of 3 adjunct Lactobacillus spp., were evaluated at d 1, 6, 30, 60, and 90 of ripening. Cheese samples (approximately 20 g) were powdered with liquid nitrogen and homogenized using water and organic solvents, and the water-soluble components were separated. A 3-μL aliquot of the extract was applied onto a reflective microscope slide, vacuum-dried, and analyzed by infrared microspectroscopy. The infrared spectra (900 to 1,800 cm⁻¹) produced specific absorption profiles that allowed for discrimination among different cheese samples. Cheeses manufactured with adjunct cultures showed more uniform and consistent spectral profiles, leading to the formation of tight clusters by pattern-recognition analysis (soft independent modeling of class analogy) as compared with cheeses with no adjuncts, which exhibited more spectral variability among replicated samples. In addition, the soft independent modeling of class analogy discriminating power indicated that cheeses were differentiated predominantly based on the band at 1,122 cm⁻¹, which was associated with S-O vibrations. The greatest changes in the chemical profile of each cheese occurred between d 6 and 30 of warm-room ripening. The band at 1,412 cm⁻¹, which was associated with acidic AA, had the greatest contribution to differentiation, indicating substantial changes in levels of proteolysis during warm-room ripening in addition to propionic acid, acetic acid, and eye formation. A high-throughput infrared microspectroscopy technique was developed that can further the understanding of biochemical changes occurring during the ripening process and provide insight into the role of adjunct nonstarter lactic acid bacteria on the complex process of flavor development in cheeses.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

新疆哈族奶酪中产蛋白酶非发酵剂乳酸菌的筛选及其系统发育分析

以采集10份新疆塔城地区哈萨克族牧民自制的奶酪为原料,用改良的ROGOSA和MRS两种培养基对其分离纯化,共筛选出43株菌种,其中球菌32株,杆菌11株。对筛选出的菌株进行产蛋白酶和菌株自溶能力测定,结合生理生化特性和16S rDNA序列同源分析和建立系统发育树分析,对挑选出的10株优势菌进行了菌株分子生物学鉴定,鉴定结果表明：菌株R6-6自溶度最高,为42.32%;而自溶度最低的菌株为R4-4,达到6.21%;菌株R2-2、R4-2、R4-7为表皮葡萄球菌（Staphylococcus epidermidis）;菌株R3-5、R10-6为乳酸片球菌（Pediococcus acidilactici）;菌株R3-2为戊糖片球菌（Pediococcus pentosaceus）;菌株R6-6为干酪乳杆菌（Lactobacillus casei）;菌株R9-5、R9-6为鼠李糖乳杆菌（Lactobacillus rhamnosus）。其中产蛋白酶的最优势非发酵剂乳酸菌（NSLAB）菌株为乳酸片球菌（P. acidilactici）。     

Cheddar干酪附属发酵剂筛选及其应用

为了筛选能够促进Cheddar干酪成熟,从泡菜、腐乳及不同产地的Cheddar中筛选具有高肽酶活力和自溶度的乳杆菌作为Ched-dar干酪附属发酵剂,并应用于Cheddar干酪的制作中.结果表明,从美国Cheddar干酪中筛选出1株Lactobacillus plantarum OD具有较高的肽酶活力和自溶度,运用于Cheddar千酪的制作中能显著提高其游离氨基酸浓度,改善其风味,有利于加快干酪的成熟.     

新疆特色干酪中乳酸菌的分离鉴定

从新疆不同牧区采集工艺不同的干酪制品,对其中的乳酸菌进行分离纯化、生理生化性质试验和16S rRNA分析.结果表明,分离、纯化出的104株乳酸菌种,有82株为乳杆菌属(Lactobacillus),12株为肠球菌属(Enterococcus),10株为魏斯氏菌属(Weissella).利用16S rRNA序列同源分析和系统发育树分析对具有不同生理生化特性的代表菌株进行了分子鉴定,鉴定结果为TNM-2与干酪乳杆菌(Lactobacillus casei)、Y5-4与食窦魏斯氏菌(Weissella cibaria)、NS2-2与植物乳杆菌(Lactobacillus plantarum)的同源性达到100％,NM-2与瑞士乳杆菌(Lactobacillus helveticus)、Y1-1与马乳酒乳杆菌(Lactobacillus kefiranofaciens)、WG与耐久肠球菌(Enterococcus durans)的同源性达到99％.     

Production of low-fat Chanco cheese using homogenized milk and adjunct lactic culture

The objective of this study was to determine the effect of the partial homogenization of milk and the use of an adjunct lactic culture on the processing, quality and yield of low-fat Chanco cheese.
  Treatments were: T1 normal-fat control and T2 low-fat control. The low-fat treatments were: T3 using homogenized milk, T4 with adjunct lactic culture and T5 both variables.
   It was concluded that the homogenization slightly increased the time of rest after cutting of the curd, and the addition of adjunct lactic culture increased the acidity of whey during a second agitation. Nevertheless, the total time and pH at the end of the process were normal and without significant statistical difference ( P  > 0.05) between treatments. When using homogenized milk the losses of fat in whey decreased by 50% with respect to the low-fat control and nearly 80% in relation to the full fat control. Consequently, the yield was increased. The physical and chemical characteristics of the Chanco cheese were not affected by the studied variables. With respect to the sensory evaluation, significant differences in colour and aroma did not appear, but there were statistically significant differences (P <  0.05) in taste, hardness, adhesiveness, cohesiveness and elasticity between the full-fat control and all the low-fat treatments. In the case of general acceptance, the treatment with adjunct lactic culture was similar to the full-fat control. In addition, it showed the least difference (mild) in elasticity and taste in relation to this control .     

Fate of Listeria innocua during production and ripening of smeared hard cheese made from raw milk

The fate of 2 different Listeria innocua strains was analyzed during the production and ripening of smeared raw milk Greyerzer cheese (Gruyère). These strains were used as surrogates for the pathogenic Listeria monocytogenes, as they are physiologically very similar. Bacterial cells were added to the cheese milk at levels of 10⁵ cfu/mL. During the first 24 h of cheese making, the number of the test strains decreased to a level of below 10² cfu/g. Obviously, the cooking temperature of 56°C and the subsequent slight temperature decrease to 50°C within 70 min contributed to a distinct reduction of Listeria counts. The counts in the cheese cores did not exceed 10³ cfu/g within 12 wk of cheese ripening and Listeria was not detectable after 24 wk. In contrast to the cores of the cheeses of the 4 batches in this study, their rinds always contained a high listerial load of approximately 10⁶ to 10⁸ cfu/g throughout the entire ripening period. The smeared surface showed an increase of pH to alkaline values, corresponding to smear microbiota development. Coryneforms and Staphylococcus counts were stable at >10⁷ cfu/cm² over 175 d, whereas yeast counts decreased to about 10⁵ cfu/cm² at the end of ripening. The study shows that the smear culture had no noticeable anti-listerial potential. When removing the rind or portioning such smeared cheese loaves with a cutting device, a postprocess contamination of the core might occur, thus presenting a major hygienic risk.     

The biodiversity and evolution of lactic flora during ripening of the Iranian semisoft Lighvan cheese

Ninety‐five isolated strains of Lactic acid bacteria (LAB) were identified from Lighvan cheese. The LAB evolution showed the dominance of lactococci and lactobacilli in the first stage and substitution of these genera by enterococci at the end of ripening. The most predominant strains were Enterococcus faecium (22.44%), Lactococcus lactis ssp. lactis (20.4%), Lactobacillus plantarum (18.36%) and E. faecalis (14.28%), respectively. Eleven and 51 different carbohydrate fermentation profiles were observed according to API 20 STREP and API 50 CH, respectively. API 20 STREP dendogram showed identical fermentation profiles of some E. faecalis and E. faecium strains, indicating that these strains might be well adapted to the whole cheese manufacture.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Use of human lysozyme transgenic goat milk in cheese making: effects on lactic acid bacteria performance

Genetically engineered goats expressing elevated levels of the antimicrobial enzyme lysozyme in their milk were developed to improve udder health, product shelf life, and consumer well-being. The purpose of this study was to evaluate the effect of lysozyme on the development of lactic acid bacteria (LAB) throughout the cheese-making process. Raw and pasteurized milk from 7 lysozyme transgenic goats and 7 breed-, age-, and parity-matched nontransgenic controls was transformed into cheeses by using industry methods, and their microbiological load was evaluated. The numbers of colony-forming units of LAB were determined for raw and pasteurized goat milk, whey, and curd at d 2 and at d 6 or 7 of production. Selective plating media were used to enumerate lactococcal species separately from total LAB. Although differences in the mean number of colony-forming units between transgenic and control samples in raw milk, whey, and cheese curd were non-significant for both total LAB and lactococcal species from d 2 of production, a significant decrease was observed in both types of LAB among d 6 transgenic raw milk cheese samples. In pasteurized milk trials, a significant decrease in LAB was observed only in the raw milk of transgenic animals. These results indicate that lysozyme transgenic goat milk is not detrimental to LAB growth during the cheese-making process.     

传统发酵酸牛奶中乳酸菌多态性分析

以从内蒙古呼伦贝尔市牧区采集的1份传统发酵酸牛奶样品为研究对象,对其进行乳酸菌的分离鉴定。通过传统纯培养法分离出17株菌,并对17株菌进行16SrDNA序列分析、多位点pheS序列分析和生理生化鉴定,鉴定的结果为11株乳酸乳球菌乳酸亚种、1株格式乳球菌、1株粪肠球菌、2株植物乳杆菌植物亚种及2株弯曲乳杆菌。乳酸乳球菌乳酸亚种为优势菌(占总分离菌株的64.7%)。