Effect of a bacteriocin‐producing strain of Lactobacillus paracasei on the nonstarter microflora of Cheddar cheese

In this study, the growth of chloramphenicol‐resistant bacteriocin‐sensitive indicator strain Lactobacillus casei DPC 2048^CM was evaluated in Cheddar cheese made with bacteriocin‐producing Lactobacillus paracasei DPC 4715. No suppression of growth of the indicator strain was observed in the cheese during ripening, and no bacteriocin production by L. paracasei DPC 4715 was detected by the well diffusion method in cheese and cheese extracts. The bacteriocin produced by L. paracasei DPC 4715 was sensitive to chymosin and cathepsin D, and it may have been hydrolysed by the rennet used for cheese manufacture or by indigenous milk proteases.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Optimal growth of Lactobacillus casei in a Cheddar cheese ripening model system requires exogenous fatty acids

Flavor development in ripening Cheddar cheese depends on complex microbial and biochemical processes that are difficult to study in natural cheese. Thus, our group has developed Cheddar cheese extract (CCE) as a model system to study these processes. In previous work, we found that CCE supported growth of Lactobacillus casei, one of the most prominent nonstarter lactic acid bacteria (NSLAB) species found in ripening Cheddar cheese, to a final cell density of 10(8) cfu/mL at 37°C. However, when similar growth experiments were performed at 8°C in CCE derived from 4-mo-old cheese (4mCCE), the final cell densities obtained were only about 10(6) cfu/mL, which is at the lower end of the range of the NSLAB population expected in ripening Cheddar cheese. Here, we report that addition of Tween 80 to CCE resulted in a significant increase in the final cell density of L. casei during growth at 8°C and produced concomitant changes in cytoplasmic membrane fatty acid (CMFA) composition. Although the effect was not as dramatic, addition of milk fat or a monoacylglycerol (MAG) mixture based on the MAG profile of milk fat to 4mCCE also led to an increased final cell density of L. casei in CCE at 8°C and changes in CMFA composition. These observations suggest that optimal growth of L. casei in CCE at low temperature requires supplementation with a source of fatty acids (FA). We hypothesize that L. casei incorporates environmental FA into its CMFA, thereby reducing its energy requirement for growth. The exogenous FA may then be modified or supplemented with FA from de novo synthesis to arrive at a CMFA composition that yields the functionality (i.e., viscosity) required for growth in specific conditions. Additional studies utilizing the CCE model to investigate microbial contributions to cheese ripening should be conducted in CCE supplemented with 1% milk fat.     

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.     

Impact of nisin producing culture and liposome-encapsulated nisin on ripening of Lactobacillus added-Cheddar cheese

This study aimed to evaluate the effects of incorporating liposome-encapsulated nisin Z, nisin Z producing Lactococcus lactis ssp. lactis biovar. diacetylactis UL719, or Lactobacillus casei-casei L2A adjunct culture into cheese milk on textural, physicochemical and sensory attributes during ripening of Cheddar cheese. For this purpose, cheeses were made using a selected nisin tolerant cheese starter culture. Proteolysis, free fatty acid production, rheological parameters and hydrophilic/hydrophobic peptides evolution were monitored over 6 mo ripening. Sensory quality of cheeses was evaluated after 6 mo. Incorporating the nisin-producing strain into cheese starter culture increased proteolysis and lipolysis but did not significantly affect cheese rheology. Liposome-encapsulated nisin did not appear to affect cheese proteolysis, rheology and sensory characteristics. The nisinogenic strain increased the formation of both hydrophilic and hydrophobic peptides present in the cheese water extract. Sensory assessment indicated that acidic and bitter tastes were enhanced in the nisinogenic strain-containing cheese compared to control cheese. Incorporating Lb. casei and the nisinogenic culture into cheese produced a debittering effect and improved cheese flavor quality. Cheeses with added Lb. casei and liposome-encapsulated nisin Z exhibited the highest flavor intensity and were ranked first for sensory characteristics.     

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.     

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。     

Impact of Autolytic and Proteolytic Lactobacilli and Nisin-Producing Culture on Proteolysis and Sensory Characteristics in Cheddar Cheese

ABSTRACT: Cheddar cheeses were made using a nisin-tolerant starter culture with either Lactobacillus delbrueckii subsp. bulgaricus UL12 (autolytic strain), Lactobacillus casei subsp. casei L2A (proteolytic strain), Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 (nisin producer), or of Lb. bulgaricus UL12 and Lc. diacetylactis UL719. Lb. bulgaricus UL12 produced more trichloroacetic acid-soluble nitrogen than did Lb. casei L2A, which produced more phosphotungstic acid-soluble nitrogen than did Lc. diacetylactis UL719. High-performance liquid chromatography analyses showed that either lactobacilli or Lc. diacetylactis UL719 increased the hydrophilic and hydrophobic peptide contents. Cheeses containing both Lb. bulgaricus UL12 and Lc. diacetylactis UL719 had the most intense old Cheddar cheese flavor after 6 mo of ripening.     

Impact of Using Exopolysaccharides (EPS)-Producing Strain on Qualities of Half-Fat Cheddar Cheese

Yield, textural, proteolysis, melting, and sensory properties of exopolysaccharide-producing Lactobacillus paracasei on properties of half-fat (about 16 g fat/100 g cheese) Cheddar cheese during ripening at 8℃ for up to six months were investigated. The results revealed that B-3 cheese, made with 2.0% (v/v) high yield exopolysaccharide-producing L. paracasei in combination with 0.011% (w/w) commercial Cheddar culture (B-3 cheese), had a 10.15, 7.71, and 10.04% separately increase in moisture content and had a 7.70, 5.05, and 6.76% separately increase in yield compared with B-2, B-4, and B-5 cheese, texture and melting characteristics were significantly improved (P < 0.05), sensory score surpassed B-4 and B-5 cheese and was similar to the full-fat one. Any differences of B-3 cheese detected among half-fat Cheddar cheeses were attributed to the presence of high yield exopolysaccharide-producing L. paracasei.     

Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts

Hard cheeses (Kefalotyri-like) were manufactured from caprine milk with yoghurt as a starter (A), and with its partial replacement with the probiotic adjuncts Lactobacillus rhamnosus LC 705 (B) and/or Lactobacillus paracasei ssp. paracasei DC 412 (C). Both adjuncts retarded the growth of enterococci, and the environment in cheese B did not favour the recovery of lactic acid bacteria (LAB) on Rogosa agar. However, better recovery of the LAB population on M17 agar from cheeses B and C made with adjuncts was recorded early in ripening, and this was accompanied by a greater decrease in pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein demonstrated that cheese C, made with Lb. paracasei ssp. paracasei as adjunct, is a better vehicle for delivery of live probiotic cells (10 ⁷   cfu/g) to the gastrointestinal tract than cheese B, made with Lb. rhamnosus ; the latter did not belong to the predominant microflora of one out of the two B cheeses. Urea-PAGE electrophoresis results indicated that adjunct lactobacilli enhanced the degradation of both α _S -casein (α _S -CN) and β-casein (β-CN). In the fresh cheese, hydrolysis of α _S -CN was more rapid than β-CN, and the free amino acid content of B and C was higher than in A. Lipolysis products were also higher in B and C than in A as ripening progressed, and the organoleptic characteristics of these cheeses resulted in higher scores, in the order C > B > A. Thus, making Kefalotyri-like cheese from caprine milk with probiotic lactobacilli, particularly Lb. paracasei ssp. paracasei, as adjunct can be considered an effective way of producing a cheese with a large number of probiotic cells.     

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。     

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.     

Viability of commercial probiotic cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in cheddar cheese

Six batches of cheddar cheese were manufactured containing different combinations of commercially available probiotic cultures from three suppliers. Duplicate cheeses contained the organisms of each supplier, a Bifidobacterium spp. (each supplier), a Lactobacillus acidophilus (2 suppliers), and either Lactobacillus casei, Lactobacillus paracasei, or Lactobacillus rhamnosus. Using selective media, the different strains were assessed for viability during cheddar cheese maturation over 32 weeks. The Bifidobacterium sp. remained at high numbers with the three strains being present in cheese at 4 x 10(7), 1.4 x 10(8), and 5 x 10(8) CFU/g after 32 weeks. Similarly the L. casei (2 x 10(7) CFU/g), L. paracasei (1.6 x 10(7) CFU/g), and L. rhamnosus (9 x 10(8) CFU/g) strains survived well; however, the L. acidophilus strains performed poorly with both decreasing in a similar manner to be present at 3.6 x 10(3) CFU/g and 4.9 x 10(3) CFU/g after 32 weeks. This study indicates that cheddar cheese is a good vehicle for a variety of commercial probiotics but survival of L. acidophilus strains will need to be improved.     

Preparation and properties of probiotic cheese high in conjugated linoleic acid content

The development of probiotic Ras cheese rich in conjugated linoleic acid (CLA) was investigated using probiotic Lactobacillus casei and Lactobacillus acidophilus starters. The cheeses were assessed for composition, proteolysis, fatty acids and fat stability, and microbiology during 3 months of ripening. The cheese made with Lb. casei and Lb. acidophilus retained high counts of the probiotic strains (～log 8) throughout storage. Ripening changes followed the normal pattern of this type of cheese during ripening. Ras cheese made with Lb. casei and Lb. acidophilus contained the highest CLA content (0.84% after 3 months) as compared to control and cheese fat had acceptable oxidative stability.     

Incorporation of Lactobacillus casei in Iranian ultrafiltered Feta cheese made by partial replacement of NaCl with KCl

Probiotic Iranian ultrafiltered Feta cheese was produced from ultrafiltration of milk with a volumetric concentration factor of 4.5:1. The heat-treated retentates were inoculated with 10(7) cfu of Lactobacillus casei LAFTI L26/mL. A mesophilic-thermophilic mixed culture of Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Streptococcus thermophilus was also used. Three percent (wt/wt) salt with different ratios of NaCl:KCl (100% NaCl, 50% NaCl:50% KCl, 75% NaCl:25% KCl, and 25% NaCl:75% KCl) were used in cheese formulation. The viability of L. casei was determined in treatments during the ripening period (90d at 5°C) within 15-d intervals. The pH, titratable acidity, and redox potential changes were monitored throughout the mentioned period. The mean pH drop rate, mean acidity increase rate, and mean redox potential increase rate were calculated at the end of the storage period. Also, total nitrogen, water-soluble nitrogen, lactic acid, and acetic acid concentrations, and syneresis and sensory characteristics of the product were measured during the mentioned period every 30d. The maximum viability of L. casei was observed within d 15 to 30 of the ripening period in the treatment containing the lowest amount of sodium. Addition of KCl enhanced syneresis. Cheeses with NaCl alone and with only 25% replacement by KCl have the highest sensory acceptability.     

Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality

To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States. DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms. According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species. This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S. thermophilus 32%, and Lactococcus 4%. Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production. Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese. All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening. The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese. The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains. These results show that certain adventitious NSLAB positively contribute to flavor development.     

模拟胃肠液环境下Mozzarella干酪提取液抗氧化活性

为评价干酪成熟过程中形成的活性肽及存活的发酵剂菌种对人体的抗氧化作用,对不同菌种组合Mozzarella干酪的蒸馏水、pH4.6醋酸缓冲液和12%三氯乙酸（TCA）提取液在模拟胃肠液环境下对DPPH自由基的清除率以及还原力进行测定。结果表明,所有不同菌种组合Mozzarella干酪的不同溶剂提取液在模拟胃肠液的环境下具有一定的抗氧化活性,并且模拟肠液的抗氧化活性大大低于模拟胃液环境,活性下降最高达88.35%。添加干酪乳杆菌（Lactobacillus casei）LPC-37的3号Mozzarella干酪提取液在模拟胃肠液环境下的抗氧化活性高于其他Mozzarella干酪,其水、pH4.6醋酸缓冲液、12%TCA提取液在模拟肠液环境下对DPPH清除率分别为73.96%、30.28%、70.27%,还原力分别为0.37、0.18、0.18。     

Accelerated Maturation of Cheddar Cheese: Microbiology of Cheeses Supplemented with Lactobacillus casei subsp. casei L2A

Growth of lactic acid bacteria (LAB) and lactobacilli was studied in Cheddar cheeses supplemented with live and heat-shocked Lactobacillus casei subsp. casei L2A and with Neutrase® to accelerate maturation. Bacterial counts of treated cheeses rapidly reached maximal values within 1 wk, whereas the control cheese reached comparable values only after 2 mo. Addition of 1.0% heat-shocked lactobacilli led to an excellent quality Cheddar cheese with a 50% increase in flavor development, as determined by sensory evaluation, compared to control cheese. Addition of Neutrase (1 × 10^-5 AU/g cheese) permitted a gain of an additional 10% while addition of higher concentrations (2 and 4 × 10^-5 AU/g cheese) resulted in undesirable bitterness.     

钾盐替代和附属发酵剂对切达干酪品质的影响

为明确降低钠含量和添加在脱脂乳中表现出具有较高血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制率的Lactobacillus paracasei M3作为附属发酵剂对切达干酪理化性质和体外抗高血压潜力的影响,将部分钾盐替代和L. paracasei M3单独和混合加入到干酪与空白组作对照,以主要成分、微生物、蛋白水解度、质构和风味为理化指标和以ACE抑制率为体外抗高血压潜力指标。结果表明,4 组干酪的主要成分差异不大（P＞0.05）,钾盐替代组、L. paracasei M3组、钾盐替代和L. paracasei M3混合组干酪在乳酸菌总数、pH 4.6可溶性氮、总游离氨基酸上显著高于空白组干酪（P＜0.05）,但是降低了干酪的硬度、pH值和有明显的苦味（P＜0.05）,而在体外抗高血压潜力方面,3 组干酪在成熟6 个月之后ACE抑制率达65.2%、74.3%和78.7%,比空白组分别高19.6%、36.2%和44.4%,由此可知,在切达干酪中钾盐替代和添加L. paracasei M3有助于具有抗高血压潜力的ACE抑制肽的产生,但是后续需要对风味进行补偿性研究。     

瑞士乳杆菌对契达干酪成熟期间所产ACE抑制肽的影响及其消化稳定性

为明确瑞士乳杆菌对契达干酪中血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制肽活性的影响,以蛋白质水解度和ACE抑制率为指标,与干酪乳杆菌组、鼠李糖乳杆菌组和空白组干酪进行对照,研究瑞士乳杆菌对干酪成熟期间蛋白质水解及ACE抑制活性的影响,并对ACE抑制活性最高时期的干酪进行消化稳定性研究。结果表明：成熟期间,3 组益生菌干酪的活菌数无明显差异（P＞0.05）,但均高于空白组;益生菌干酪的蛋白质水解程度和ACE抑制活性显著高于空白组（P＜0.05）,其中瑞士乳杆菌干酪的蛋白质水解程度最强,活性最高（79.71%）。模拟消化后,瑞士乳杆菌干酪活菌数降低14.30%,ACE抑制活性显著增加（P＜0.05）,达到86.06%,多肽质量浓度增加至2.81 mg/mL;研究不同分子质量超滤组分消化后的ACE抑制活性发现,其中大于10 kDa的多肽活性升高,小于10 kDa的活性下降。此外,添加瑞士乳杆菌不影响干酪的整体可接受性。因此,瑞士乳杆菌能促进干酪ACE抑制肽的产生并提高其活性,消化后活性的升高主要与大分子肽的降解有关。