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.     

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。     

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.     

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.     

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.     

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.     

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％.     

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%)。     

Biodiversity of Bacteriocin-Producing Lactic Acid Bacteria from Mexican Regional Cheeses and their Contribution to Milk Fermentation

The aim of this work was to examine the biodiversity of bacteriocin-producing lactic acid bacteria from homemade cheeses produced in Veracruz (México) and assess their contribution as adjunct cultures in dairy products. Ninety-three presumptive bacteriocinogenic strains were detected by direct antagonism assays and 29 of them were active against Enterococcus faecalis NRRL-B537, Listeria innocua 062 AST, or Listeria monocytogenes ATCC19115 by the well diffusion test using cell-free supernatants, adjusted to pH 6.0 to exclude inhibition by organic acids. Positive isolates were identified by partial sequencing of the 16s rDNA as Pediococcus acidilactici (four isolates), Enterococcus faecium (17 isolates), Lactobacillus plantarum (six isolates) and Lactobacillus fermentum (two isolates). RAPD-PCR discriminated seven groups with a 50% similarity and revealed the presence of the same isolates. The coding genes for the synthesis of plantaricin EF, plantaricin JK, plantaricin N, plantaricin NC8 and the inducing peptide plantaricin A were detected by PCR in L. plantarum. Similarly, enterocin P and pediocin PA-1 genes were amplified from Enterococcus and Pediococcus genomic DNA, respectively. Overall, co-culturing of bacteriocin producing Lactobacillus and Pediococcus strains with the dairy starter Lactococcus lactis IPLA947 did not interfere with milk acidification. Lactose consumption, acidification rate and production of lactic acid were unchanged. Nonetheless, higher levels of acetic acid, ethanol and succinic acid were detected depending on the strain. Our results demonstrate the diversity of bacteriocinogenic species in homemade Mexican cheeses which may be used as adjunct cultures to enhancing safety of this well-appreciated cheese while providing a richer range of metabolites.     

Classification of Swiss cheese starter and adjunct cultures using Fourier transform infrared microspectroscopy

The acceptability of Swiss cheese largely depends on the flavor profile, and strain variations of cheese cultures will affect the final quality. Conventional biochemical methods to identify the cultures at the strain level are time-consuming and expensive, and require skilled labor. Our objective was to develop rapid classification methods of starter cultures at the strain level by using a combination of hydrophobic grid membrane filters and Fourier transform infrared (FTIR) spectroscopy. Forty-four pulsed-field gel electrophoresis-verified strains of starter and nonstarter cultures including Streptococcus thermophilus, Propionibacterium freudenreichii, and Lactobacillus spp. were analyzed. The strains were grown on their respective agar media, transferred to broth media, and incubated. Then, cultures were centrifuged and the pellets were resuspended in saline solution (10 μL). Aliquots (2 μL) of the suspended bacterial solution were placed onto a grid of the hydrophobic grid membrane filters, having 6 grids per each strain analyzed. The dried filters were read by FTIR microspectroscopy fitted with an attenuated total reflectance probe. Collected spectra were statistically analyzed by a soft independent modeling of class analogy (SIMCA) for pattern recognition. Classification models were developed for Streptococcus thermophilus, Propionibacterium freudenreichii, and Lactobacillus spp. strains. The models showed major discrimination in the spectral region from 1,200 to 900 cm⁻¹ associated with signals from phosphate-containing compounds and various polysaccharides in the cell wall. The developed method allowed for rapid classification of several Swiss cheese starter and nonstarter cultures at the strain level. This information provides a detailed overview of microbiological status, which would enable corrective measures to be taken early in the cheese making process, limiting production of inferior quality cheese and minimizing defects. This method could be an effective tool to identify and monitor activity of cheese and other dairy starter cultures.     

活性乳酸菌功能饮料的研制

活性乳酸菌乳饮料是近年发展起来的兼营养、保健功能为一体的新型乳饮料,产品中含有大量的活性乳酸菌,具有助消化和调整胃肠功能等功效。研究以发酵酸奶为主要原料,通过正交实验筛选最优配方,其结果为酸奶含量为35%、蔗糖用量9%、酸奶香精用量为0.4%、薄荷香精用量为0.035%。酸果奶稳定剂0.25%,果胶0.1%;产品酸度70～75°T;通过分析检测,产品活乳酸菌数为4.1×106cfu/mL。产品符合国家质量卫生标准,且香甜爽口,组织细腻、均匀。     

Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese

Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains).     

乳酸菌肉品发酵剂的选择

从发酵肉制品中分离、纯化乳酸菌132株,对它们的主要发酵特性进行了测定,以生产干发酵香肠誓前篓,从中选出4株乳酸菌优选菌株R17、P20、P30和J33。4株菌生长速度有显著差异,温度对其生长速度有显著的影响。其中R17和P20之间没有拮抗关系,可以作为混合发酵剂。P30和J33可以作为单菌发酵剂。     

New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography–mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health.     

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.