Nucleotide sequencing,purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.     

Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing,purification, and characterization

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.     

Lactobacillus casei expressing methylglyoxal synthase causes browning and heterocyclic amine formation in Parmesan cheese extract

Undesired browning of Parmesan cheese can occur during the latter period of ripening and cold storage despite the relative absence of reducing sugars and high temperatures typically associated with Maillard browning. Highly reactive α-dicarbonyls such as methylglyoxal (MG) are products and accelerants of Maillard browning chemistry and can result from the microbial metabolism of sugars and AA by lactic acid bacteria. We demonstrate the effects of microbially produced MG in a model Parmesan cheese extract using a strain of Lactobacillus casei 12A engineered for inducible overexpression of MG synthase (mgsA) from Thermoanaerobacterium thermosaccharolyticum HG-8. Maximum induction of plasmid-born mgsA led to 1.6 mM MG formation in Parmesan cheese extract and its distinct discoloration. The accumulation of heterocyclic amines including β-carboline derivatives arising from mgsA expression were determined by mass spectrometry. Potential MG-contributing reaction mechanisms for the formation of heterocyclic amines are proposed. These findings implicate nonstarter lactic acid bacteria may cause browning and influence nutritional aspects of Parmesan by enzymatic conversion of triosephosphates to MG. Moreover, these findings indicate that the microbial production of MG can lead to the formation of late-stage Maillard reaction products such as melanoidin and β-carbolines, effectively circumventing the thermal requirement of the early- and intermediate- stage Maillard reaction. Therefore, the identification and control of offending microbiota may prevent late-stage browning of Parmesan. The gene mgsA may serve as a genetic biomarker for cheeses with a propensity to undergo MG-mediated browning.     

Lactobacillus demonstrate thiol-independent metabolism of methylglyoxal: Implications toward browning prevention in Parmesan cheese

Endogenous production of α-dicarbonyls by lactic acid bacteria can influence the quality and consistency of fermented foods and beverages. Methylglyoxal (MG) in Parmesan cheese can contribute toward undesired browning during low temperature ripening and storage conditions, leading to the economic depreciation of affected cheeses. We demonstrate the effects of exogenously added MG on browning and volatile formation using a Parmesan cheese extract (PCE). To determine the influence of Lactobacillus on α-dicarbonyls, strains were screened for their ability to modulate concentrations of MG, glyoxal, and diacetyl in PCE. It was found that a major metabolic pathway of MG in Lactobacillus is a thiol-independent reduction, whereby MG is partially or fully reduced to acetol and 1,2-propanediol, respectively. The majority of lactobacilli grown in PCE accumulated the intermediate acetol, whereas Lactobacillus brevis 367 formed exclusively 1,2-propanediol and Lactobacillus fermentum 14931 formed both metabolites. In addition, we determined the inherent tolerance to bacteriostatic concentrations of MG among lactobacilli grown in rich media. It was found that L. brevis 367 reduces MG exclusively to 1,2-propanediol, which correlates to both its ability to significantly decrease MG concentrations in PCE, as well as its significantly higher tolerance to MG, in comparison to other lactobacilli screened. These findings have broader implications toward lactobacilli as a viable solution for reducing MG-mediated browning of Parmesan cheese.     

Short communication: Evidence for methylglyoxal-mediated browning of Parmesan cheese during low temperature storage

Brown pigmentation can occasionally form in Parmesan cheese during the ripening process, creating an unappealing appearance and associated off-flavors. The browning reactions proceed at refrigerated temperatures and in the relative absence of reducing sugar, contrary to typical Maillard browning. Residual sugars, lipid oxidation products, byproducts of fermentation, and (or) enzymes may react with primary amines in cheese to form brown pigmentation and concomitantly generate volatile compounds unique to each of these reactions. Determining the volatile profiles provides a means of understanding the potential substrates involved and causative reaction pathways. This work is divided into 2 segments. The first portion characterized the pigmentation and the browning index of Parmesan cheeses with and without extensive browning. The second phase examined the volatile character of the cheeses using gas chromatography-mass spectrometry. Various pyrazines, such as 2,3,5-trimethylpyrazine and 3,5-diethyl-2-methylpyrazine, were found in the brown cheeses but were not present in the white samples. The formation of pyrazines is proposed to result from the spontaneous condensation of aminoacetone. Aminoacetone can be formed by the Strecker degradation of amino acids with methylglyoxal, the latter a byproduct of sugar fermentation. Evidence is provided to support a browning pathway mediated by the production of methylglyoxal in Parmesan cheese.     

Fate of Listeria monocytogenes during the manufacture and ripening of Parmesan cheese

Parmesan cheese was made from a mixture of pasteurized whole and skim milk that was inoculated to contain ca. 10(4) to 10(5) cells of Listeria monocytogenes/ml. Curd was cooked at 51 degrees C (124 degrees F) for ca. 45 min. During cheese making, maximum numbers of L. monocytogenes appeared just before cooking; at this point, the increase over initial numbers was a .61 to 1.0 order of magnitude. During cooking of curd, the average decrease in numbers of L. monocytogenes was a .22 order of magnitude. During cheese ripening, numbers of L. monocytogenes decreased almost linearly and faster than reported for other hard cheeses. Listeria monocytogenes strain California died faster than did strain V7. Listeria monocytogenes were not detected in cheese after 2 to 16 wk of ripening, depending on the strain of the pathogen and the lot of cheese. Parmesan cheese made in this study was not a favorable medium for survival of L. monocytogenes.     

Study of organic acids, volatile fraction and caseins of a new Halloumi-type cheese during ripening in whey brine

Organic acids, fat hydrolysis, volatile compounds and sensory characteristics of a new brine cheese which combines characteristics of Halloumi and Feta cheeses during its ripening in whey brine (100g NaCl L⁻¹) were studied. Thermotolerant protease of Mucor miehei as a coagulant enzyme and a mixture of thermotolerant starter cultures Enterococcus faecium 0165 (0.5% w/w) and Lactobacillus casei 80 10D were used. Good quality new Halloumi-type cheese was produced with higher proteolysis than traditional Halloumi cheese kept in whey brine. The volatile compounds identified comprised alcohols, aldehydes, ketones, acids, esters, hydrocarbons and sulphur compounds. Ethanol was the dominant volatile compound determined. Lactic acid was the dominant acid produced; its concentration increased during ripening, reaching a maximum value of 9929 mg kg⁻¹ at day 30. Acetic acid was also found in high amounts, which increased during cheese ripening. Lipolysis of cheese was not intense. The most abundant acids of the mature cheese were palmitic, oleic and acetic acid. The Halloumi-type cheese scored higher in the sensory analysis when fresh than did the mature cheese.     

Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

瑞士乳杆菌对契达干酪成熟期间品质的影响

干酪的成熟是形成干酪特有的组织状态、质地和风味的关键工序。将分离自内蒙古传统乳制品中的瑞士乳杆菌SMN2-1作为非发酵性乳酸菌添加到契达干酪的生产中,通过检测其成熟过程中理化指标、气味变化和质构特性等指标,分析了瑞士乳杆菌对契达干酪成熟的影响。结果显示:在90 d的成熟期内,两组干酪的蛋白质、脂肪、水分和盐分含量之间差异不显著(p>0.05),但添加瑞士乳杆菌SMN2-1的干酪成熟第90 d的气味明显改变,内聚性、弹性和咀嚼性等物性指标均高于对照,因此添加瑞士乳杆菌SMN2-1可以有效促进蛋白质分解和干酪的成熟,同时改善干酪的风味和质地。     

益生菌切达干酪成熟过程中细菌群落的多样性分析

采用选择性培养基和聚合酶链式反应和变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gelelectrophoresis,PCR-DGGE）技术,研究益生菌切达干酪成熟过程中（6 ℃,180 d）细菌群落构成及益生菌（干酪乳杆菌LC2W）的存活情况。结果表明：SBM和MSE等选择性培养基存在选择专一性不强的缺点,不能客观反映干酪内各种微生物的动态变化;随着切达干酪成熟时间的增加,发酵剂嗜热链球菌和乳酸乳球菌的数量明显下降,而非发酵剂菌群乳杆菌的数量和主要种类呈上升趋势;干酪成熟180 d后,干酪乳杆菌LC2W的存活量仍高于1×108CFU/g。切达干酪能作为干酪乳杆菌LC2W存活的良好载体;PCR-DGGE技术和选择计数法联用更加适合干酪细菌群落结构的分析。     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

Strains of the Lactobacillus casei group show diverse abilities for the production of flavor compounds in 2 model systems

Cheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification.     

Characterization of microflora in homemade semi-hard white Zlatar cheese

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.     

Microbiological study of Manura, a hard cheese made from raw ovine milk in the Greek island Sifnos

Changes in the microbial flora of Manura, a raw ovine milk cheese, were studied during ripening. In general, the various microbial groups developed better on the cheese surface than in the interior, but red wine treatment had an inhibitory effect on their growth and microbial counts decreased ( P  < 0.05) more rapidly on the cheese surface than in the interior. NaCl and moisture of the cheese affected microbial levels significantly. Thus, Enterobacteriaceae and coliforms were reduced sharply ( P  < 0.05) during ripening on a straw bed (∼3 months) and they were not detected in mature cheese. Lactic acid bacteria predominated over the other microbial groups throughout ripening. Leuconostoc mesenteroides ssp. cremoris , Pediococcus pentosaceus and Lactobacillus paracasei ssp. paracasei , frequently found in maturing cheese, could be used as starters to make this cheese. Moreover, the lactic acid bacteria predominating in mature cheese, such as Weissella paramesenteroides , Lactobacillus bifermentans and Lactobacillus brevis , may contribute to cheese ripening through their biochemical activities.     

Manufacture of low-fat Cheddar cheese by exopolysaccharide-producing Lactobacillus plantarum JLK0142 and its functional properties

This study aimed to evaluate the effect of exopolysaccharide (EPS)-producing Lactobacillus plantarum JLK0142 on the ripening characteristics and in vitro health-promoting benefits of low-fat Cheddar cheese. Three batches of cheese were made by employing a non-EPS–producing cheese starter (control), in combination with Lb. plantarum JLK0142 as an adjunct and the purified EPS as an ingredient. Lactobacillus plantarum JLK0142 survived well in cheese, with counts of 7.99 log cfu/g after 90 d of ripening. All experimental cheeses (with adjunct culture or EPS ingredient) had higher moisture, proteolysis, and sensory scores, and lower hardness and cohesiveness compared with the control cheese. Water-soluble extracts from the experimental cheeses outperformed that of the control in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl radicals, and inhibiting α-amylase, angiotensin-converting enzyme, and HT-29 tumor cell growth. Therefore, incorporation of the EPS-producing culture of Lb. plantarum JLK0142 is promising for improvement of low-fat cheese quality and bioactivities.     

Esterases of lactic acid bacteria and cheese flavour: Milk fat hydrolysis,alcoholysis and esterification

Free fatty acids (FFAs) and esters derived from FFAs are important flavour compounds in cheese. Evidence is provided that esterases of lactic acid bacteria (LAB) catalyse not only hydrolysis of milk fat glycerides to release FFAs, but also synthesis of esters from glycerides and alcohols via a transferase reaction. The esterases of LAB prefer di- and monoglycerides for both hydrolysis and ester synthesis and are, in fact, alcohol acyltransferases that use both water (hydrolysis) and alcohol (alcoholysis) as acyl acceptors. Therefore, esterases of LAB can impact on both the lipolytic and ester flavours of cheese. The impact of esterases of LAB on cheese flavour can be controlled by manipulating the amount of the esterase, by regulating alcohol availability and/or by increasing the mono- and diglyceride composition of milk fat. In addition, esterification may play a role in ester synthesis in hard cheeses (e.g. Italian type) where water activity is low.     

Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.     

Influence of adjuncts as a debittering aids in encountering the bitterness developed in cheese slurry during accelerated ripening

An attempt was made to accelerate the flavour development in cheese base with the help of exogenous proteolytic and lipolytic enzymes (1:1 proportion, each at the rate of 0.025% by weight of cheese‐base) and ripening at elevated temperatures (i.e. 20 ± 1 °C) for up to 12 days. To counter the bitterness developed, adjunct cultures were used: viable or attenuated (freeze‐shocked or heat shocked). Study of biochemical characteristics, electrophoretic pattern and sensory evaluation of the product were carried out. An acceptable enzyme‐modified, lightly salted cheese base was obtained using 0.025% each of proteolytic and lipolytic enzymes, along with 5% starter culture and adjuncts followed by ripening up to 12 days. Freeze‐shocked adjunct Lactobacillus helveticus produced enzyme‐modified cheese base with no detectable bitterness. The usage of exogenous enzymes, temperature of ripening, ripening period and interactions amongst these parameters had significant (P < 0.01) influence on all of the biochemical characteristics monitored.     

基于快速成熟模型的藏灵菇发酵切达干酪挥发性风味物质分析

通过建立快速成熟干酪模型,采用固相微萃取法提取传统藏灵菇发酵的切达干酪模型与商品发酵剂制作的切达干酪模型中挥发性成分,并结合气相色谱-质谱联用技术和气相色谱-嗅闻技术对萃取成分进行鉴定,结果表明醇类和酯类是藏灵菇发酵切达干酪成熟过程中的主要风味物质。藏灵菇发酵切达干酪模型中风味物质的种类和含量都明显高于商业发酵剂制作的切达干酪模型,其中酯类物质的变化最为显著。感官评价和风味分析结果表明,藏灵菇发酵切达干酪模型中酯类和醇类物质种类和含量更为丰富,风味更强,水果香味更浓郁,还具有酒香味。     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.