Hot topic: Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation

Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.     

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.     

Comparison of the compositional, microbiological, biochemical, and volatile profile characteristics of nine Italian ewes' milk cheeses

Nine Italian ewes’ milk cheeses were compared for compositional, microbiological, biochemical, and volatile profile characteristics. Mean values for the gross composition were rather similar among cheeses. The lowest pH values were found for cheeses that used primary starters. At the end of ripening, cheeses made from raw milk contained >6.0 log₁₀ cfu/g of nonstarter lactic acid bacteria. Several species of lactobacilli were identified, but Lactobacillus plantarum and Lactobacillus paracasei were dominant. Random amplified polymorphic DNA-PCR analysis showed the biodiversity among the strains, and in several cases a relationship with the cheese of provenance. Cheeses differed mainly for secondary proteolysis, as shown by the principal component analysis applied to reversed-phase fast protein liquid chromatography data of the pH 4.6-soluble fractions and by determination of the free AA. A total of 113 volatile components were identified in the Italian Pecorino cheeses by solid-phase microextraction coupled with gas chromatography-mass spectrometry analysis. The volatile profiles of the 9 cheeses differed significantly. Quantitatively, alcohols were the most abundant chemical class for some cheeses, whereas ketones were the most abundant for other cheeses. Esters and carboxylic acids were largely found. Specific volatile components seemed to distinguish specific cheeses.     

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.     

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.     

A factory-scale application of secondary adjunct cultures selected from lactic acid bacteria during Puzzone di Moena cheese ripening

The lactic acid populations of 2 seasonal Puzzone di Moena cheeses made from winter and summer raw cow's milk were characterized at different ripening times. Lactic acid bacteria (LAB) were isolated on selective media and subjected to genetic typing and identification. The species most frequently found during ripening were Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus. The different strains recognized by random amplification of polymorphic DNA-PCR were characterized for their acidifying and proteolytic activities to select nonstarter LAB to be used as secondary adjunct cultures (SAC). For each of the 3 above species, a strain showing weak acidification and high proteolytic capacity was selected. The 3 strains (Lb. paracasei ssp. paracasei P397, Lb. plantarum P399, and P. pentosaceus P41) constituted a mixed SAC used at 2 levels of concentration (10³ and 10⁴ cfu/mL) in experimental cheese making at dairy factory-scale. The analysis of volatile organic compounds as well as sensory analyses showed that the preferred level of SAC inoculation was 10³ cfu/mL.     

发酵香肠中分离纯化的三株乳酸菌产酸特性研究

为了研究从发酵香肠中分离纯化的3株乳酸菌粪肠球菌(Enterococcus faecalis)、戊糖片球菌(Pediococcus pentosaceus)和肠膜明串珠菌(Leuconostoc mesenteroides)的产酸性能,对这3株菌在不同pH、温度、NaCl、NaNO₂条件下的生长情况及产酸情况进行了测定。研究结果显示:这3株菌中,肠膜明串珠菌和戊糖片球菌的生长特性较好;粪肠球菌的生长特性虽不如肠膜明串珠菌和戊糖片球菌,但产酸能力最好,戊糖片球菌耐盐性最好、肠膜明串珠菌耐亚硝酸盐的特性最好;在不同温度和pH条件的测试中,肠膜明串珠菌的生长能力最好,粪肠球菌次之。这3株乳酸菌在发酵肉制品中均产生乳酸。总之,这3株菌均具有用于发酵制备乳酸的能力。     

麦芽表面乳杆菌的筛选及其产酸特性的初步研究

从多种大麦、商品麦芽和绿麦芽表面筛选出96株乳杆菌,最终筛选得到1株菌被鉴定为发酵乳杆菌。对其在麦汁中的产酸性能进行了初步研究,发酵温度为48℃,12 h产酸能达到0.48%。该菌培养液在50%分割剩余量下,14 h能恢复到分割前的酸度;在90%分割剩余量下,6 h能达到起始酸度。     

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.     

抑制活菌泡菜贮藏过程中品质劣变的研究

目的:研究活菌泡菜产品在贮藏流通过程中由微生物引起的产品品质变化,开发延长泡菜贮藏期的新方法。方法:对从泡菜中分离鉴定出来的8种乳酸菌,在不同pH培养基条件下检测各种乳酸菌生长速度。对8种乳酸菌进行产气测定。在发酵前添加泡菜汁、嗜酸乳杆菌、植物乳杆菌、木糖醇等,在不同温度条件下发酵和贮藏,对泡菜的色泽进行评分,对酸度、混浊度、挥发性物质进行测定。结果:在pH 4.5时,植物乳杆菌是优势菌,其次是短乳杆菌。在pH 4.0~3.0时,嗜酸乳杆菌是优势菌,其次是植物乳杆菌,主要产气菌是短乳杆菌、假肠膜明串珠菌、坏发酵乳杆菌。pH 4.0~4.5时,主要产气菌是短乳杆菌。在pH 3.5时,主要是坏发酵乳杆菌产气。在pH 3.0时,不容易产气。发酵前添加2 mL/L 1×105 CFU/mL植物乳杆菌和20 mg/g木糖醇,可显著延长常温和低温下泡菜的贮藏期和货架期。结论:1×105 CFU/mL植物乳杆菌和20 mg/g木糖醇处理,显著抑制常温贮藏泡菜的总挥发物积累,抑制泡菜的异硫氰酸烯丙酯、萘类积累,有利于维持原初气味特征。     

乳酸菌细菌素的高效表达方法研究

乳酸菌细菌素作为天然的生物防腐剂,因其高效、无毒等特点而备受关注,但目前只有乳酸链球菌素实现了工业化生产。乳酸菌细菌素产量低是限制其大规模生产的瓶颈之一。从高产菌株筛选、发酵条件调控、基因工程技术、诱导调控和胁迫刺激应答5个方面,综述了提高乳酸菌细菌素产量的方法,期望为提高乳酸菌细菌素产量的研究提供新思路。     

酸奶粉生产技术的研究进展

酸奶粉具有丰富的营养价值,在医疗和食品工业中得到了广泛的应用。酸奶粉既保持了酸奶原有的营养价值和功能特性,又延长了货架期、降低了成本,因此具有广阔的发展前景。从酸奶粉的干燥方法、再水合以及保护剂等角度阐述了酸奶粉的生产工艺,并进行了展望。     

乳酸菌的科学与技术

叙述了过去一个世纪以来有关乳酸菌的科学与技术发展概况，包括乳酸菌的发现、分离和分类体系的确立。介绍了乳酸菌的生态和共存关系中的作用以及乳酸菌生成物的利用，乳酸菌育种之技术与理论的发展，并提出了今后展望及建议对乳酸菌的研究内容。     

异型乳酸菌2种常用鉴定方法的比较

选用18株乳酸菌和1株标准菌株植物乳杆菌1.2158(Lactobacillus plantarum1.2158)进行葡萄糖产酸产气实验和hot-loop实验,以探讨异型乳酸菌两种常用鉴定方法的异同。结果如下:(1)葡萄糖产酸产气法的检测结果表明,实验菌株产酸能力不同。实验菌株中有7个形成明显的琼脂柱位移,鉴定为异型乳酸菌,占总菌株量的36.8%;(2)hot-loop法检测结果表明有13个菌株形成气泡或柱状泡泡,鉴定为异型乳酸菌,占总菌株量的68.4%;(3)菌株L-6、L-14、L-15、L-16、L-18和L-19为葡萄糖产酸产气法和hot-loop法共同检测出的异型乳酸菌株;L-5为葡萄糖产酸产气法检测出的特异异型乳酸菌株;L-1、L-2、L-3、L-7、L-8、L-9和L-17为hot-loop法检测出的特异异型乳酸菌株。hot-loop法具高灵敏度,但葡萄糖产酸产气法具观察菌株产酸能力的方便性和判断菌株产气的直观性;葡萄糖产酸产气法和hot-loop法检测的异型乳酸菌株有互补性。将两种方法进行结合应用,会使鉴定结果更具有有效性和快速性。     

肉制品发酵剂的筛选与发酵特性的研究

以MRS培养基为基础培养基,从自然发酵香肠和自制腌肉中分离、筛选出三株乳酸菌(F1、F2、F3),对其生化特性、发酵特性、生长状况、产酸能力、致死温度及菌株间的拮抗性进行研究,结果表明:3株乳酸菌均符合肉制品发酵剂的要求,可以作为肉制品发酵剂;菌株F1和F3,F2和F3可以作为肉制品的混合发酵剂.     

传统泡菜中高产酸乳酸菌的筛选与鉴定

以湖南地区家庭自制50余年泡菜液为样品,采用纯培养方法分离纯化得到乳酸菌。对分离得到的乳酸菌进行溶血活性检验,产酸性能测定,并研究高产酸菌株的耐酸耐胆盐、自聚集疏水性、抗氧化、降解亚硝酸盐、生长特性及生物安全性研究。结果表明,从泡菜样品中分离出来70株乳酸菌,检验出完全无溶血活性乳酸菌42株;筛选出高产酸性能乳酸菌9株,编号为JT1-31、JT1-21、JT1-12、JT1-25、JT1-6、JT1-16、JT1-34、JT3-23、JT3-10,通过16S rDNA鉴定,均为植物乳植杆菌（Lactiplantibacillus plantarum）,菌株JT1-12耐酸性最好,pH值3.0时存活率为156.85%;菌株JT3-10耐胆盐能力最好,0.3%胆盐浓度存活率为145.04%;菌株JT1-16自聚集和疏水性最高,分别为66.13%和44.19%;菌株JT1-21的DPPH清除率最高,为51.93%;菌株JT1-25的α-葡萄糖苷酶抑制率最高,为20.36%;9株菌株亚硝酸盐降解率都较高,在90%以上,清除率最高的菌株是JT1-12,清除率为94.98%,通过产生物胺鉴定,9株菌株均不产生物胺。该研究表明,从传统泡菜中分离的9株植物乳植杆菌具有良好的益生潜能,为发掘食品源乳酸菌提供参考和依据。     

双层包埋乳酸菌及乳酸菌素之应用

天然乳酸菌如果未经改造,生命力十分脆弱,是绝对无法耐久存.乳酸菌专业厂商塞尔生技公司投入可观资源,成功开发出一种独特的双层包埋技术在天然乳酸菌表面包覆蛋白质,然后再包一层特殊胶体,解决了长久以来一直无法突破的天然乳酸菌不易保存与不耐胃酸的问题.利用这种特殊双层包埋技术,最外层的胶体在强酸性的胃酸中凝结,保护乳酸菌.等到益生菌进入较高酸碱值较高的十二指肠时,胶体自然分解溶化,释放益生菌,而后第二层的蛋白质就会促进乳酸菌与肠壁纤毛附着,同时提供乳酸菌繁殖所需营养.利用双层包埋技术包埋的乳酸菌,稳定性及再加工性远优于一般未经包埋的乳酸菌,除了传统应用在酸奶、奶粉或胶囊型、铝箔包型保健食品等一般常见的乳酸菌商品上之外,也已成功运用在果汁、糖果、巧克力、饼干、口香糖、冰淇淋等食品及锭剂型保健食品上.除了可以有效延长储架期间的活性达到6月以上,还可充分扩张益生菌之应用范围.利用乳酸菌代谢产物生产功能特殊的乳酸菌素,也是乳酸菌厂商积极开发的项目之一,其耐热性更强,用量较低, 具有针对性, 不会误伤有益微生物. 目前也成功开发出多种可应用在食品、化妆品及保健品方面的乳酸菌素,功能包括食品保鲜、抗幽门罗杆菌、抗痤疮杆菌等.安全可靠, 使用方便.     

发酵肉制品中乳酸菌的主要发酵特性

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如耐盐性、耐硝性、产黏液、产气等进行了测定,并比较了它们在不同培养基中的产酸情况。