Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.     

Microbiological and physicochemical characterization of small-scale cocoa fermentations and screening of yeast and bacterial strains to develop a defined starter culture

Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.     

Fermentation studies of stored cocoa beans

Acetic and lactic acid bacteria on fermented cocoa beans were maximally 2.0×10⁶ and 1.9×10⁶ c.f.u./g wet wt, respectively. Acetic and lactic acids were detected on the second and fourth days of fermentation and were maximally 140 and 45 mg/10 g beans, respectively. There was a positive correlation between the sizes of the relevant microbial populations and the amounts of acids produced during fermentation.     

The cocoa bean fermentation process: from ecosystem analysis to starter culture development

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro‐organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well‐fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp‐bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa‐derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.     

Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.     

新疆喀什地区母乳中乳酸菌的多样性分析

【目的】对新疆喀什地区母乳中乳酸菌多样性进行分析。【方法】采用菌落培养、显微镜观察、Repetitive genomic fingerprinting(Rep-PCR)指纹图谱和16S r RNA基因序列分析相结合的方法研究母乳中乳酸菌的菌群分布。【结果】从11份母乳中共分离出乳酸菌193株,利用16S r RNA基因序列同源分析和系统发育树对代表菌株进行了分子鉴定,193株乳酸菌隶属于4个属,分别为Lactobacillus(22株)、Streptococcus(42株)、Lactococcus(40株)、Enterococcus(89株)。其中,Enterococcus所占比例最大,达到46%。【结论】新疆喀什地区母乳中乳酸菌多样性丰富,极具开发潜力,将为开发母乳中的益生菌以及安全可靠的微生态制剂提供理论依据。     

新疆传统发酵乳品中乳酸菌与 酵母菌的益生特性

目的观察新疆传统发酵乳品中分离的14种菌株的生长特点及产酸能力,筛选出具有较强耐胆盐能力,并能在人工胃肠液中存活的菌株。方法对10株乳酸菌和4株酵母菌进行生长曲线、pH、耐胆盐能力和耐人工胃肠液检测。结果 10株乳酸菌和4株酵母菌具有良好的生长曲线和产酸能力;马乳酒样乳杆菌具有较强的耐胆盐能力;希氏乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工胃液能力;乳酸乳球菌、哈尔滨乳杆菌、瑞士乳杆菌、马乳酒样乳杆菌、乙醇假丝酵母和东方伊萨酵母具有较强的耐人工肠液能力。结论 10株乳酸菌和4株酵母菌具有优良的益生特性,有望成为益生菌制剂的备用菌株。     

Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2

We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S.?cerevisiae W303-1A and in the isogenic mutants jen1?, ady2? and jen1? ady2?, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells.     

Lactic Acid Utilization by the Cutaneous Micrococcaceae

Human cutaneous staphylococci and micrococci utilized lactic acid as an energy source on a minimal medium. Propionic acid was not utilized, but l(+)-lactic acid and pyruvic acid could replace ld-lactic acid as a substrate. Selected strains of cocci were inhibited more by the l(+) and d(-) forms of lactic acid than the balanced ld form, particularly at pH 5.6. With proper dilution of substrate, lactic acid was utilized by selected strains in the presence of 10 mug of oleic and palmitic acids per ml.     

rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers

Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.     

新疆伊犁地区原牛乳中乳酸菌的多样性分析

【目的】对新疆伊犁地区原牛奶中乳酸细菌的遗传多样性进行分析。【方法】采用菌落培养、Rep-PCR(Repetitive genomic fingerprinting)指纹图谱和16S r RNA基因序列分析相结合的方法研究牛乳内乳酸菌的遗传多样性。【结果】从5份原牛乳中分离出乳酸菌29株,基因序列分析和系统进化分析显示29株乳酸菌隶属于5个属,分别为:Lactococcus、Lactobacillus、Leuconostoc、Pediococcus和Enterococcus。优势属为Leuconostoc(27.6%),其次为Lactococcus(24.0%)。【结论】新疆伊犁地区原牛乳中乳酸菌多样性丰富,为开发新疆地区益生乳酸菌提供了丰富的活性资源。     

Cellular Fatty Acid Profiles of Lactobacillus and Lactococcus Strains in Relation to the Oleic Acid Content of the Cultivation Medium

Cellular fatty acids of 10 strains of lactic acid bacteria were analyzed. The purpose of this work was to find lactic acid bacteria with high lactobacillic acid contents. The bacteria studied were unable to synthesize oleic acid. Some strains did not synthesize lactobacillic acid, although all were able to form dihydrosterculic acid. Twenty-one to thirty-four percent of the fatty acid content of Lactobacillus fermentum and L. buchneri was lactobacillic acid, and these species were chosen for future studies of environmental factors affecting cyclopropane fatty acid synthesis.     

Screening of microorganisms for biodegradation of poly(lactic-acid) and lactic acid-containing polymers.

The ability of some microorganisms to use lactic acid stereocopolymers and copolymers with glycolic acid as sole carbon and energy sources was studied under controlled or natural conditions. First, 14 filamentous fungal strains were tested in liquid cultures, adopting total lactic acid consumption, nitrogen source exhaustion, and maximal biomass production as selection criteria. Two strains of Fusarium moniliforme and one strain of Penicillium roqueforti were able to totally assimilate DL-lactic acid, partially soluble racemic oligomers (MW = 1,000), and the nitrogen source. Only one strain of F. moniliforme was able to grow on a poly(lactic acid)-glycolic acid copolymer (MW = 150,000) after 2 months of incubation at 28 degrees C on synthetic agar medium. Mycelium development was examined by scanning electron microscopy. F. moniliforme filaments were observed to grow not only at the copolymer surface but also through the bulk of the copolymer. In a second approach, plates made of a racemic poly(lactic acid) were buried in the soil before being incubated in petri dishes containing mineral agar medium under controlled conditions. Five strains of different filamentous fungi were isolated, and their ability to assimilate racemic poly(lactic acid) oligomers was tested in liquid cultures.     

Characterization and determination of origin of lactic acid bacteria from a sorghum-based fermented weaning food by analysis of soluble proteins and amplified fragment length polymorphism fingerprinting

The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains were Lactobacillus plantarum strains, 47 were Leuconostoc mesenteroides strains, 25 were Lactobacillus sake-Lactobacillus curvatus strains, 17 were Pediococcus pentosaceus strains, 13 were Pediococcus acidilactici strains, and 7 were Lactococcus lactis strains. L. plantarum and L. mesenteroides strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.