Dissertations / Theses on the topic 'Bacteria producing lactic acid'

Lactic acid bacteria were isolated from 10 samples of the traditionally fermented foods (5 samples of Vietnamese fermented pork roll and 5 samples of the salted field cabbage) and 5 samples of fresh cow milks collected from households in Vietnam. 22 strains of lactic acid bacteria were isolated for inhibition to Lactobacillus plantarum JCM 1149. Of these, only 2 strains including DC1.8 and NC1.2 have rod shape, the others have coccus shape. 7 strains showing higher antibacterial activity were selected for checking spectrum of antibacteria with indicator bacteria consistting of Bacillus subtilis ATCC 6633, Enterococcus faecium JCM 5804 and Staphylococcus aureus TLU. By which, 3 strains including NC3.5 (from Vietnamese fermented pork roll), DC1.8 (from salted field cabbage) and MC3.19 (from fresh cow milk) were selected because of their higher antibacterial ability. However, the antibacterial activity of the lactic acid bacteria can be based on their disposable compounds and some other antibacterial compounds produced during their growth (such as lactic acid, H2O2, bacteriocins, etc.). For seeking lactic acid bacteria with capability of producing bacteriocins, antibacterial compounds with protein nature, 3 above strains were checked sensitiveness to proteases (including protease K, papain, α – chymotrypsin and trypsin). Because bacteriocins are proteinaceous antibacterial compounds, so their antibacterial activity will be reduced if proteases are added. The result showed DC1.8 and MC3.19 were capable of producing bacteriocin during culture process. They were identified as Lactobacillus acidophilus and Lactococcus lactis and classified, respectively, based on analysis chemical characterisitcs by standard API 50 CHL kit and phylogeny relationship by 16s rRNA sequences.
Các chủng vi khuẩn lactic được phân lập từ 10 mẫu thực phẩm lên men truyền thống (5 mẫu nem chua, 5 mẫu dưa cải bẹ muối) và 5 mẫu sữa bò tươi được thu thập từ các hộ gia đình ở Việt Nam. 22 chủng vi khuẩn lactic đã được phân lập với tiêu chí có khả năng kháng lại vi khuẩn kiểm định Lactobacillus plantarum JCM 1149. Trong số đó, 2 chủng DC1.8 và NC1.2 có tế bào hình que, các chủng còn lại có tế bào hình cầu. 7 chủng thể hiện hoạt tính kháng khuẩn cao được lựa chọn để xác định phổ kháng khuẩn rộng hơn với ba loài vi khuẩn kiểm định Bacillus subtilis ATCC 6633, Enterococcus faecium JCM 5804 và Staphylococcus aureus TLU. Từ đó lựa chọn được 3 chủng có hoạt tính kháng khuẩn cao hơn hẳn. Các chủng này gồm NC3.5 phân lập từ nem chua, DC1.8 phân lập từ dưa cải bẹ muối và MC3.19 phân lập từ sữa bò tươi. Tuy nhiên, hoạt tính kháng khuẩn của vi khuẩn lactic bao gồm những hợp chất nội tại có trong nó và cả những hợp chất được sinh ra trong quá trình phát triển của nó (như axit lactic, H2O2, bacteriocin, …). Với định hướng tìm chủng vi khuẩn lactic có khả năng sinh bacteriocin, chất kháng khuẩn có bản chất protein, 3 chủng trên được kiểm tra độ nhạy cảm với các protease (gồm protease K, papain, α – chymotrypsin và trypsin). Do bacteriocin là chất kháng khuẩn có bản chất protein nên hoạt tính kháng khuẩn của chúng sẽ bị giảm nếu protease được bổ xung vào. Kết quả lựa chọn được chủng DC1.8 và MC3.19 có khả năng sinh bacteriocin. Hai chủng này được phân loại đến loài nhờ vào phân tích đặc điểm sinh hóa bằng kit API 50 CHL và mối quan hệ di truyền thông qua trình tự gen 16s rRNA. Kết quả phân loại đã xác định chủng DC1.8 thuộc loài Lactobacillus acidophilus và chủng MC3.19 thuộc loài Lactococcus lactis.

Submitted by JESSELEINE CRISTINE MONTEIRO DA SILVA null (jesse_cris@hotmail.com) on 2017-09-20T20:56:09Z No. of bitstreams: 1 Dissertação final Jesseleine.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5)
Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-27T17:48:51Z (GMT) No. of bitstreams: 1 silva_jcm_me_araiq.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5)
Made available in DSpace on 2017-09-27T17:48:51Z (GMT). No. of bitstreams: 1 silva_jcm_me_araiq.pdf: 1516064 bytes, checksum: 56e50bf83bbdb465716b33121512f13f (MD5) Previous issue date: 2017-09-06
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Devido ao crescente aumento de doenças transmitidas por alimentos, a segurança microbiológica se torna uma questão de saúde pública pelas suas características de endemicidade, alta morbidade e pela dificuldade da adoção de medidas de controle desses microrganismos. Diante deste fato, o objetivo deste trabalho foi sintetizar e caracterizar os análogos peptídicos LeuB e LeuC-1 derivados de bacteriocinas naturais denominadas Leucocinas. Os peptídeos foram sintetizados manualmente pelo método de síntese em fase sólida, submetidos à desproteção total e clivagem, com liberação dos peptídeos brutos. Foram realizadas as análises comparativas usando HPLC e ESI-MS, e com os respectivos peptídeos puros foram feitos os ensaios antimicrobiano, enzimático, permeabilização, antioxidante, hemolítico e de espectroscopia de dicroísmo circular. Com isso, observou-se que o método de síntese dos análogos foi adequado e o processo de purificação possibilitou a obtenção dos peptídeos com alto grau de pureza. O peso molecular teórico dos peptídeos foi confirmado por espectrometria de massas. O LeuB apresentou uma maior capacidade em inibir o crescimento de Escherichia coli O157:H7 e em Salmonella sorovar Typhimurium, enquanto que LeuC-1 apresentou efeito de inibição de crescimento de Listeria monocytogenes e também de S. sorovar Typhimurium. É importante destacar que todas essas bactérias são de interesse na área de alimentos, já que são as causadoras da maioria dos casos de infecção alimentar. O ensaio de inibição enzimática com DNA girase e Topoisomerase IV mostrou que apenas o peptídeo LeuB possui capacidade de inibição destas enzimas bacterianas, sugerindo um possível mecanismo de ação deste derivado de Leucocina. Este peptídeo também apresentou a capacidade de permeabilizar miméticos de membrana bacteriana composto de POPC/POPG (75/25). Por sua vez, o peptídeo LeuC-1 não apresentou capacidade significativa de inibição da atividade das enzimas DNA girase e Topoisomerase IV e também não apresentou capacidade de permeabilização de miméticos de membrana. Porém, LeuC-1 apresentou uma boa atividade antioxidante, obtida pelo método de ABTS. Ambos os peptídeos apresentaram baixa toxicidade em eritrócitos, comprovadas pelos ensaios hemolíticos. Estruturalmente, os peptídeos LeuB e LeuC-1 tendem a se estruturar em α-hélice. Assim sendo, este trabalho possibilitou a obtenção de dois peptídeos com potencial de aplicação como conservantes alimentares a partir de mecanismos distintos de ação sem apresentar citotoxicidade para células vermelhas do sangue. LeuB possui uma suposta atuação como inibidor de topoisomerases bacterianas e capacidade de permeabilização de miméticos de membrana. LeuC-1 possivelmente atua em diferentes vias metabólicas da bactéria, porém ainda não foi possível elucidar o mecanismo alvo deste mimético peptídico.
Due to the increase of foodborne diseases, microbiological safety becomes a public health issue due to its characteristics of endemicity, high morbidity and the difficulty of adopting control measures of these microorganisms. In view of this fact, the objective of this work was to synthesize and characterize LeuB and LeuC-1 peptidics analogues derived from natural bacteriocins called Leucocins. The peptides were synthesized manually by the solid phase synthesis method, subjected to total deprotection and cleavage, with release of the crude peptides. Comparative analyzes were performed using HPLC and ESI-MS, and with the respective pure peptides the antimicrobial, enzymatic, permeabilization, antioxidant, hemolytic and circular dichroism spectroscopy. With this, it was observed that the method of synthesis of the analogs was adequate and the purification process allowed to obtain the peptides with high purity. The theoretical molecular weight of the peptides was confirmed by mass spectrometry. LeuB showed a greater capacity to inhibit the growth of Escherichia coli O157: H7 and Salmonella serovar Typhimurium, whereas LeuC-1 presented inhibition effect of growth of Listeria monocytogenes and also of S. serovar Typhimurium. It is important to highlight that all these bacteria are interesting in the area of food, since they are the cause of most cases of food infection. The enzyme inhibition assay with DNA gyrase and Topoisomerase IV showed that only the LeuB peptide has the ability to inhibit these bacterial enzymes, suggesting a possible mechanism of action of this Leucocin derivative. This peptide also showed the ability to permeabilize bacterial membrane mimetics composed of POPC/POPG (75/25). On the other hand, the LeuC- 1 peptide did not present significant capacity to inhibit the activity of the enzymes DNA gyrase and Topoisomerase IV and also did not present permeabilization capacity of membrane mimetics. However, LeuC-1 presented a good antioxidant activity, obtained by the ABTS method. Both peptides had low erythrocyte toxicity, as demonstrated by hemolytic assays. Structurally, the LeuB and LeuC-1 peptides tend to be α-helix structured. Therefore, this work enabled two peptides with application potential as food preservatives to be obtained from distinct mechanisms of action without presenting red blood cell cytotoxicity. LeuB has a supposed action as inhibitor of bacterial topoisomerases and permeabilization capacity of membrane mimetics. LeuC-1 possibly acts on different metabolic pathways of the bacterium, but it has not yet been possible to elucidate the target mechanism of this peptidic mimetic.
CNPq: 150928/2015-7

The glycopeptide antibiotics vancomycin and teicoplanin are used to treat infections caused by Gram positive bacteria. The formation of nascent peptidoglycan chains and cross linking of the cell wall is inhibited because the drugs bind specifically to the D-alanyl-D-alanine portion of the pentapeptide chain in peptidoglycan precursors. Plasmid-mediated, high-level resistance to both antibiotics in Enterococcus sp. is associated with production of a novel D-alanine:D-alanine (D-Ala:D-Ala) ligase of altered substrate specificity. This enzyme, VanA, synthesises the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac), which is incorporated into cell wall precursors, instead of D-Ala-D-Ala. Vancomycin has a 1000 fold lower affinity for cell wall precursors terminating in the hydroxyacid. VanA and other plasmid-borne van genes essential for high-level glycopeptide resistance in enterococci lie within the inverted repeats of a transposon; Tn1546, which has a distinctly different G+C ratio to enterococcal DNA, suggesting an exogenous origin. Lactic acid bacteria such as Lactobacillus sp. and Leuconostoc sp. are intrinsically resistant to glycopeptide antibiotics. Analysis of their cell wall precursors reveals that they terminate in D-Lac, suggesting a similar mechanism of resistance to that of the enterococci. The mechanism of cell wall synthesis in vancomycin-sensitive and resistant lactic acid bacteria and VanA-type enterococci was investigated. The D-Ala:D-Ala ligase from the glycopeptide-sensitive lactic acid bacterium, Lactobacillus delbrueckii, was purified directly from cell extracts and characterised. No D-Ala:D-hydroxyacid ligase activity was detected in extracts from the glycopeptide-resistant Lactobacillus brevis. Subsequently, the ligase of Leuconostoc mesenteroides (Lmddl), which had already been sequenced, was cloned and overexpressed, to allow purification and characterisation of the enzyme.

This thesis is an investigation into the production of exopolysaccharides (EPS) produced by strains of Lactobacillus delbrueckii ssp. bulgaricus and Lactococcus lactis ssp. cremoris. These are used in the dairy industry for the production of yoghurt and fermented drinks products. For many years EPS producing lactic acid bacteria have been used by the dairy industry as a thickening agent in the production of yoghurt. However, this EPS producing trait is unstable and is readily lost which can cause an alteration in the texture of the final product. It was found that all the strains of Lb. delbrueclcii ssp. bulgaricus and Le. /aetis ssp. eremoris produced quantities of EPS that could be used for further analysis. They were found to be in the molecular weight range of 6.6 x 1 06g /mol to 1.26 x 1011 glmol and were composed of different quantities of glucose, galactose and rhamnose. Temperature, carbon source and shaking all affected the quantities of EPS produced by all strains of Le. laetis ssp. eremoris. The firmness and viscosity of fermented milks produced by strains of Lb. delbrueckii ssp. bulgaricus were higher than those produced by strains of Le. laetis ssp. cremoris indicating that firmness and viscosity are not solely related to the levels of EPS production. A 40kb plasmid was found in all strains of Le. /aetis ssp. cremoris that could potentially contain the genes for EPS production. The plasmid could not be removed using elevated temperature or with the addition of acriflavin. Fourier transform infrared spectroscopy (FTIR) showed that it was possible to differentiate different strains based on their spectra and that differences were found in the protein and EPS regions of the spectra. It was also established that the age of culture, whether the growth medium was liquid or solid and the carbon source of the growth media had an effect on the FTIR spectra produced and the ability to differentiate between strains. There is further potential to develop this technique to provide a quick and easy method of identifying strains of lactic acid bacteria and monitor their EPS producing ability.

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: The presence and growth of lactic acid bacteria (LAB) in wine and their influence on wine quality has received much attention in recent years. Lactic acid bacteria are responsible for conducting malolactic fermentation (MLF) in wine. The benefits associated with malolactic fermentation in terms of deacidification of wine and the contribution to wine flavour and complexity have also recently been the topic of research. It is impossible to describe malolactic fermentation as distinctly desirable or undesirable in terms of its influence on the final quality of wine. The benefits and disadvantages are dependent upon viticultural region, grape variety, wine composition, winemaking techniques and the style and objectives of the winemaker. Brandy production is a multi-stage process in which base wine production, distillation technique and wood maturation all have a large influence on the final chemical profile and organoleptic quality of the brandy. The volatile composition of the base wine, which basically undergoes a concentration process during the subsequent double distillation phase, is critical in determining the aroma and flavour quality of the final brandy product. Thus, the brandy is only as good as the base wine it is distilled from. The aims of this study were to determine the effect of lactic acid bacteria and spontaneous malolactic fermentation on the quality of brandy base wine and the resulting distillate, and to determine which LAB species had been responsible for the occurrence of spontaneous MLF. This study showed that LAB are present at high numbers and are able to conduct spontaneous MLF of brandy base wines. It was shown that the incidence of spontaneous MLF varied from year to year. In 1998, 50% of the commercially produced base wines had undergone partial MLF prior to distillation. In 1999 and 2000 respectively, 34% and 45% of the commercial base wines had undergone partial MLF prior to distillation. The occurrence of spontaneous MLF had an influence on the chemical composition and the sensory quality of the base wine and distillate. There was an increase in the concentrations of ethyl lactate, acetic acid and diethyl succinate in samples that had undergone MLF. There was also a decrease in the concentrations of esters, such as iso-amyl acetate, ethyl acetate, ethyl caproate, hexyl acetate and 2-phenethyl acetate in these same samples. Sensory evaluation of the base wines and distillates demonstrated that samples that had undergone MLF differed significantly from samples that had not undergone MLF. It was also shown that distillates that had not undergone MLF had a slightly better aroma profile than those that had. Sweet aromas, like chocolate and caramel, as well as negative aromas, like chemical or solvent, were more prominent in brandy distillates that had undergone MLF. Herbaceous and fruity aromas were more intense in distillates not having undergone MLF. Fifty-four strains, all Gram-positive and catalase negative, were isolated at different stages of brandy production. Seven strains were isolated from the grape juice, 15 strains were isolated from the base wine, 20 strains were isolated during MLF and 12 strains were isolated from the base wine after MLF had been completed. Based on C02 production from glucose and gluconate, 17 strains were classified as facultatively heterofermentative and 37 strains as obligately heterofermentative. Fifteen of the 37 obligately heterofermentative strains were rod-shaped and were regarded as lactobacilli. The remaining 22 strains were oval or cocci-bacilli shaped. The isolates were identified to species level by using numerical analysis of the total soluble cell protein patterns, 16S rRNAsequencing and polymerase chain reaction (PCR) with species-specific primers. The facultative heterofermentative lactobacilli were identified as Lactobacillus paracasei and Lactobacillus p/antarum. The fifteen obligately heterofermentative lactobacilli were identified as members of the species Lactobacillus brevis, Lactobacillus verrniforme, Lactobacillus buchneri and Lactobacillus hi/gardii. The 22 obligate heterofermentative isolates, with a coccoid morphology, could be grouped into two clusters and were identified as Oenococcus oeni. O. oeni was the species responsible for the occurrence of spontaneous MLF in most of the commercial base wines. Lb. brevis, Lb. hi/gardii and Lb. paracasei were also isolated from commercial base wines that had undergone spontaneous MLF. In nine out of 14 experimental base wine samples that had undergone spontaneous MLF, O. oeni was again the predominant species. Lb. brevis, Lb. hi/gardii and Lb. paracasei were identified in the remaining experimental base wine samples. This is the first report of the presence of Lb. perecese! and Lb. vermiforme in brandy base wine. It was shown that the occurrence of spontaneous MLF had a negative effect on the quality of brandy base wine, but that was shown to be due to the different species and strains performing MLF. In the non-preferred distillate samples, Lactobacillus spp. had performed MLF or had developed after or during MLF.
AFRIKAANSE OPSOMMING: Die teenwoordigheid en die vermoë van melksuurbakterieë (MSB) om in wyn te groei, is 'n onderwerp wat al heelwat nagevors is. Melksuurbakterieë is verantwoordelik vir die uitvoering van appelmelksuurgisting (AMG) in wyn. Die voordele verbonde aan appelmelksuurgisting, ten opsigte van die verlaging van die totale suurinhoud en die bydrae tot die verbeterde geur en kompleksiteit van die wyn, is ook al goed bestudeer. Wat die invloed op die finale wynkwaliteit betref, is dit byna onmoontlik om AMG as uitsluitlik gewens óf ongewens te beskou. Die voordele en nadele van AMG is afhanklik van verskeie faktore, nl. wingerdkundige streek, druifkultivar, wynsamestelling, wynmaakpraktyke, asook die styl en doelwitte van die wynmaker. Die produksie van brandewyn is 'n multistapproses waarin die bereidingsmetode van die basiswyn, die distillasietegniek en houtveroudering 'n groot invloed op die finale kwaliteit en chemiese samestelling van die brandewyn het. Die vlugtige verbindings van die basiswyn, wat tydens die dubbele distillasieproses gekonsentreer word, is van wesenlike belang in die bepaling van die aroma en geur van die finale brandewynproduk. Brandewyn is dus inderdaad net so goed soos die basiswyn waarvan dit gestook is. Die doelwitte van hierdie studie was om te bepaal wat die invloed van MSB en die voorkoms van spontane AMG op die kwaliteit van die basiswyn en die distillaat is, asook om die MSB wat vir die voorkoms van spontane AMG verantwoordelik was, te identifiseer. Hierdie studie het bewys dat MSB in hoë getalle teenwoordig was en dat dit in staat is om die spontane AMG van basiswyne uit te voer. Daar is bewys dat die voorkoms van spontaneAMG moontlik van jaar tot jaar kan verskil. In 1998 het 50%, in 1999 het 34% en in 2000 45% van die kommersieel-geproduseerde basiswyn gedeeltelike AMG spontaan voor distillasie ondergaan. Daar is ook gevind dat spontane AMG 'n invloed op die chemiese samestelling en sensoriese kwaliteit van die basiswyn en die distillaat gehad het. Daar was 'n toename in die konsentrasies van etiellaktaat, asynsuur en diëtielsuksinaat in monsters wat spontane AMG ondergaan het. In dieselfde monsters was daar ook 'n afname in die konsentrasies van iso-amielasetaat, etielasetaat, etielkaproaat, heksielasetaat en 2-fenielasetaat. Sensoriese evaluering van die basiswyne en distillate het getoon dat daar betekenisvolle verskille was tussen die monsters wat AMG ondergaan het en dié wat nie AMG ondergaan het nie. Daar is bewys dat die distillate wat nie AMG ondergaan het nie, 'n beter aromaprofiel gehad het as dié wat AMG ondergaan het. Soet geure, soos sjokolade en karamel, en negatiewe geure, soos "chemies" en "oplosmiddel", was prominent in distillate wat AMG ondergaan het. Kruidagtige en vrugtige geure was meer intensief in distillate wat nie AMG ondergaan het nie. Vier-en-vyftig bakteriese rasse, almal Gram-positief en katalase-negatief, is gedurende die verskillende stadia van brandewynproduksie geïsoleer. Sewe rasse is uit druiwesap, 15 rasse gedurende die alkoholiese fermentasie, 20 rasse gedurende AMG en 12 rasse na voltooiing van AMG geïsoleer. Op die basis van koolstofdioksied (C02)-produksie vanaf glukose en glukonaat is 17 rasse as fakultatief heterofermentatief en 37 rasse as obligaat heterofermentatief geklassifiseer. Vyftien van die 37 obligaat-heterofermentatiewe rasse was staafvormig en is as lactobacilli geïdentifiseer. Die oorblywende 22 het ovaal of kokkus-bacillusvormige selmorfologie getoon. Identifikasie tot op spesievlak is gedoen deur van numeriese analise van die totale oplosbare selproteïenprofiele, 16S-rRNAvolgordebepalings en spesie-spesifieke inleiers vir die polimerasekettingreaksie (PKR) gebruik te maak. Die fakultatief-heterofermentatiewe rasse is as Lactobacillus paracasei en Lactobacillus p/antarum geklassifiseer. Die 15 obligaat heterofermentatiewe stafies is as Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus hi/gardii en Lactobacillus vermiforme geïdentifiseer. Die 22 ovaal, obligaat heterofermentatiewe isolate kon in twee groepe ingedeel word en is as Oenococcus oeni geïdentifiseer. Daar is bevind dat O. oeni-isolate vir die voorkoms van spontane AMG in die meeste van die kommersiêle basiswyne verantwoordelik was. Lb. brevis, Lb. hi/gardii en Lb. paracasei is ook uit kommersiêle basiswyne wat spontane AMG ondergaan het, geïsoleer. In nege uit 14 van die eksperimentele basiswyne wat spontane AMG ondergaan het, was O. oeni die dominante spesie. In die oorblywende eksperimentele wyne is Lb. brevis, Lb. hi/gardii en Lb. paracasei aangetref. Hierdie is die eerste vermelding van die teenwoordigheid van Lb. paracasei and Lb. vermiforrne in brandewynbasiswyn. Daar is gevind dat die voorkoms van spontane AMG "n negatiewe invloed op brandewynkwaliteit het, maar dit is as gevolg van die verskeidenheid van MSB-spesies en rasse wat voorkom. In die distillate wat deur die proepaneel afgekeur is, het Lactobacillus spesies die AMG deurgevoer, of het dit tydens of na AMG ontwikkel.

Malolactic fermentation (MLF) is an important process in the wine production. MLF results from the metabolism of certain lactic acid bacteria (LAB) and consists in the conversion of malate to lactate and CO2. Except deacidification, MLF can improve the quality and microbiological stability of the wines. The aim of this project was to investigate the preservation and utilization of the LAB with particular reference to the MLF. In order to measure the effect of various preservation methods and their productivity, an assay of cell vitality was developed. This measured the capacity to overcome and recover from freezing and freeze-drying. It was shown that this method was easily used and reliable. The effect of cultural conditions on the cryotolerance and vitality of the LAB was investigated, including: (1) the growth phase, (2) the growth temperature, (3) the medium pH, (4) composition of the medium, and (5) preincubation conditions. The optimal cultural conditions to obtain higher vitality after freezing varied with the species of LAB. When the pH of culture medium was controlled at pH 5 the LAB attained the highest vitality after freezing. When L. plantarum was preincubated in 5g/l yeast extract solution at 25°C for 1 hour, the survival rate of L. plantarum greatly increased, from 5.2% to 46.5%. The conditions of freeze-drying of the LAB were investigated. It was found that 4% yeast extract was the most effective protectant for L. plantarum and L. brevis and 5% glutamate were the best protectant for O. oeni. When the LAB was frozen quickly at -65°C, the vitality obtained was higher than those frozen slowly at -20°C after freeze-drying. Another factor to be considered important was ethanol tolerance when the freeze-dried malolactic bacteria were used in wines. Among the suspension media tested, 5% glutamate and 10% sucrose were the best for freeze-dried L. brevis and O. oeni respectively to obtain high vitality in high ethanol solutions. These studies showed that there were no consistent underlying processes that could be easily identified and that preservation was a species specific, multifactorial process. The MLF was then investigated further by studying the effect of wine components on the batch MLF of L. brevis and O. oeni using a defined synthetic wine. This uniquely allowed a systematic study of the MLF in high alcohol environments. Alcohol tolerance was dependent on temperature and important fermentation intermediates such as citrate, pyruvate and malate. Malolactic fermentations were inhibited when glucose concentration was 2 g/1 to 6 g/1. The inhibition to MLF of O. oeni caused by glucose was relieved when fructose was present. Nutritional status was also an important factor that affected the MLF, when the synthetic wine did not contain added yeast extract, malic acid degradation of L brevis and O. oeni was low (6.1% and 54.3% respectively). Rapid and continuous malolactic fermentation was achieved in the membrane bioreactor (MBR) with high cell density of O. oeni (greater than 108CFU/ml). More than 95% degradation of malic acid in the synthetic wine was obtained at 0.48 1/h of flow rate and 10.4 h residence time. High ethanol concentration of wine was main factor that caused the loss in malic acid degrading activity of O. oeni in the MBR. The poor nutritional condition of wine was not the main factor causing loss in the stability of malic acid degrading activity of O. oeni. The shear stress had little influence on the malic acid degradation of O. oeni under the conditions investigated. Ethanol stress adaptation could improve the stability of malic acid degrading activity of O. oeni in the MBR.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第11617号
農博第1473号
新制||農||905(附属図書館)
学位論文||H17||N4010(農学部図書室)
UT51-2005-D366
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 清水 昌, 教授 加藤 暢夫, 教授 植田 充美
学位規則第4条第1項該当

Certaines bactéries lactiques (BL) peuvent se développer pendant le stockage des viandes et inhiber des bactéries pathogènes ou d’altération qui sont un problème dans l’industrie des produits carnés. Plusieurs mécanismes sont alors mis en jeu comme une compétition générale et/ou la production de molécules inhibitrices comme les bactériocines. Dans cette thèse, 198 souches de BL ont été testées pour leur capacité à inhiber des espèces d’altération (Brochothrix thermosphacta, Clostridium estertheticum) ou pathogènes (Listéria monocygènes, Campylobacter jejuni) susceptibles de se développer sur la viande. Parmi 7 isolats capables d’être compétitifs dans des conditions de laboratoire, 3 souches de Lactobacillus sakei et 1 souche de Carnobacterium maltaromaticum se sont avérées également efficaces vis-à-vis de certaines espèces indésirables après inoculation de viande de mouton ou de bœuf, sans affecter la qualité sensorielle des produits. En effet, la croissance de ces souches de BL était associée à l’inhibition du développement de L. Monocytogènes et C. Jejuni et à la réduction de production de gaz par C. Estertheticum. L’analyse des produits sensoriels et altérants de viande de mouton inoculée par un cocktail de 3 souches de L. Sakei à montré une augmentation de la qualité d’acides lactique et acétique et une diminution de pH n’affectant pas l’acceptabilité sensorielle de la viande. Les résultats de cette étude ont permis la caractérisation d’une collection de BL, essentiellement composée de L. Sakei pouvant servir de base pour le développement d’outils utiles à la bio-conservation de viande réfrigérée Néozélandaise destinée à l’exportation
Some lactic acid bacteria (LAB) are capable or growing on stored meat and inhibiting pathogenic and spoilage bactéria of concern to the meat industry using a range of mechanisms including general competition and/or the production of specific inhibitory molecules such as bacteriocins. In this thesis, 198 LAB strains were screened for their ability to inhibit spoilage species (Brochothrix thermophacta, Clostridium estertheticum) or pathogenic species (Listeria monocytogenes, Campylobacter jejuni) that can occur on meat. Out of 7 isolates able to compete in laboratory conditions, 3 Lactobacillus sakei and 1 Carnobacterium maltaromaticum strains were shown to be also efficient against some undesired species when seeded in lamb or beef meat, without affecting the sensory quality of the products. Indeed growth of the LAB strains was associated to the inhibition of L. Monoctytogenes and C. Jejuni and a reduction of gas production by C. Estertheticum. Sensory and spoilage product analysis of stored lamb inoculated with a 3-strain cocktail of L. Sakei showed higher levels of lactic and acetic acids and a lower pH without affecting the sensory acceptance of meat. The results of this work characterize a collection of predominantly L. Sakei LAB strains that may provide the basis for developing useful products for the bio-preservation of chilled New-Zeland export meat

Probiotics are marketed throughout the world to promote the health of the consumer by improving the microorganisms that normally occur in the intestinal tract (Tannock, 1997). It has also been suggested that probiotics can prevent pathogen infections by adhering to the intestinal mucosa (Lee, Lim, Teng, Ouwehand, Tuomola, & Salminen, 2000). While probiotics can be delivered to the infected areas in multiple fashions, microencapsulation is a newer form of delivering probiotics straight to the infected area. A whey protein microcapsule is thought to protect the probiotics from stomach acid and delivers the treatment to the affected area. To ensure this microencapsulation treatment is affective, the microcapsules will be stained and imaged to see if the microcapsules are constructed in a way which is consistent with the theory: a whey protein microcapsule surrounding bacteria and fat droplets. Through these experiments, it was shown that the microcapsule was not constructed as previously thought. Instead of a thin layer of protein surrounding the bacteria, it more closely resembled a solid ball of protein with bacteria and fat trapped inside. The bacteria are able to survive stomach like conditions (0.1M HCl for 8 hours) due to other forms of microencapsulation.

Food fermentation is a method widely used in the past to extend the storage life of food. Numerous studies on fermented food have revealed that they not only have biopreservative properties but also health benefits. Lactic acid bacteria are the major group of microorganisms involved in food fermentation and the properties that influence food are primarily due to the compounds released from microorganisms such as organic acids and bacteriocins. Their health benefits are exerted through several mechanisms including inhibiting the growth of pathogenic bacteria and modifying the host immune response. A number of strains that have been investigated show different properties even between the same species thus emphasizing the importance of strain identification. To determine if some traditional fermented Swedish foods contain lactic acid bacteria, bacteria from four fermented Swedish foods (two surströmming and two sausages) were isolated using MRS broth. Bacterial isolates were examined for their colony and cell morphology and Gram staining and were found to be predominantly Gram-positive cocci or rods. 16S rRNA PCR amplifications of selected isolates was performed using universal prokaryotic primers and sequenced. The sequencing results showed that the bacterial isolates from Oskars surströmming Filéer and Gognacs medvurst were Lactobacillus sakei and the isolate from Mannerströms surströmming was Enterococcus sp. This study showed that the traditional Swedish fermented food evaluated did contain lactic acid bacteria.

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Greek dry fermented sausages. Samples were taken at different stages, and 150 "wild' strains were isolated. The majority of the strains isolated were assigned to the species Lactobacillus plantarum biotype (1) (43.3 %) followed by Lb. curvatus, Lb. pentosus (10.7 %), Lb. brevis biotype (1) (8.7 %), Lactococcus lactis subsp. lactis biotype (1) (6.7 %) and Leuconostoc mesenteroides subsp. mesenteroid.es biotype (2) (5.3 %). The possibility of bacteriocin production was tested using the agar well diffusion assay (AWDA). One strain was found to produce bacteriocin (Leuconostoc mesenteroides E131) and its purification was attempted using precipitation with ammonium sulphate, cation exchange, and reverse phase chromatography. Moreover, the purification of curvaticin L442 was attemped, a bacteriocin produced by Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without addition of starters. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Lb sakei I154, a bacteriocin producing strain isolated from Italian fermented sausage, and the semi-purified bacteriocin from Leuconostoc mesenteroides E131 were validated via industrial trials to evaluate whether the product (fermented sausages) maintains the technological characteristics and the traditional quality characteristics. Three fermentations under controlled conditions were conducted and at the end of these fermentations, products were sliced and packaged under controlled atmosphere (80% N2 + 20% CO2) and stored at 4+/-2 °C for 12 weeks to determine the shelf life of the product. Finally, from the previous industrial trials the proper production parameters were determined as well as the most effective packaging techniques, resulting in the conduction of a Standard Operation Procedures Guide concerning the whole production of traditional fermented sausages.

碩士
國立澎湖科技大學
食品科學系碩士班
107
127 strains of lactic acid bacteria with phytase activity were screened from 614 strains of lactic acid bacteria isolated from the algae in Penghu keeping in our laboratory. Among them, 7 strains of the highest activity lactic acid bacteria were identified as 3 strains of Lactobacillus plantarum (5-1, 1.2U/mL; 20-4-1, 1.7U/mL; 62-3, 1.72U/mL) , 1 strain of Lactobacillus brevis (12-5, 1.88 U/mL), 1 strain of Lactobacillus rhamnosus (128-1, 1.24 U/mL) and 2 strains of Lactobacillus fermentum (62-6, 0.91 U/mL; 366-1, 1.37 U/mL). After fermenting with phytase producing LAB, phytic acid in brown rice pulp, shelled wheat pulp, and whole soy milk medium can be degraded effectively. The pH was significantly reduced to 5 after 12 hours of fermentation. The number of bacteria in the fermentation for 24 hours reached a maximum. The phytic acid degradation gradually slowed down after 48 hours of fermentation. Comparing the number of viable bacteria and the amount of phytic acid degradation， Lactobacillus plantarum 20-4-1 is the best strain which can be used in grain fermentation, the phytic acid residue after fermentation for 72 hours were 13.76 mg/g (36%) of whole soybeans, 2.56 mg/g of brown rice (28%), and 5.63 mg / g (50.0%) of the shelled wheat.

碩士
國立中正大學
化學工程所
94
Abstract Lactate is one of the major fermented products from lactic acid bacteria. Furthermore, diacetyl is a small amount of valuable byproduct for dairy food and pharmaceutical application. In the past decade, the reserchers only focus on doing parts of the experiments in order to understand the regulatory relationship of lactic acid bacteria metabolism and raise the productivity of diacetyl by trial and error processes. These take a lot of money and time but in vein. If employing the technology of metabolic engineering, we can make a better mutation strategy for producing diacetyl. In this study, metabolic flux analysis (MFA) and inverse flux analysis are introduced to analyze the impact of the objective flux when the other flux is slightly perturbed. In addition, we use the quadratic programming optimization to construct the physiological flux distributions of lactic acid bacteria metabolism. Further, we compared the enzyme sets of elementary flux modes with each other to characterize the functional routes and the essential route in lactic acid bacteria metabolism.

碩士
東海大學
畜產學系
88
The study was conducted to select slime-producing lactic acid bacteria for manufacturing ropy yogurt, in order to replace the addition of pectin in the traditional yogurt, and to save the cost of production and improve the quality of yogurt. Ten strains of commercial lactic acid bacteria, five modified media (MEPS-1, MEPS-2, MEPS-3, MRS plus broth and Elliker broth), four incubation temperatures (27, 32, 37 and 42℃) and three incubation times (8, 16 and 24 hours) were used in the experiments. The results showed that the thread forming property of MEPS-2 medium（9% SNF skimmilk containing 0.35% yeast extract, 0.35% peptone and 5% lactose）under 32℃ at 16 hours was significantly higher than other treatment combinations (P<0.05), so using it as the selective conditions. Twelve commercial fermented dairy products were collected and then the above selective conditions were used to select the slime-producing lactic acid bacteria. It was found that five products named Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ, had the thread forming property. After a series of isolation procedures, four slime-producing strains named A1, A2, B2 and E were purified. A1 and A2 were identified as Streptococcus thermophilus, and B2 and E were identified as Lactobacillus bulgaricus. According to the combination of rod and coccus to match A1, A2, B2 and E. The change of qualities during four-weeks storage of 10% SNF yogurt made with L+S, A1+B2 or A2+E culture incubated at 32℃ and 42℃ was determined. The results showed that, the thread forming property, viscosity , syneresis and lactic acid bacterial counts of A1+B2 and A2+E sets were significantly better than L+S set when incubating at 32℃ or 42℃ (P<0.05), and 32℃ group was better than 42℃ group. The sensory evaluation results of ropy and traditional yogurt showed that, A1+B2 and A2+E sets had lower syneresis, higher viscosity, smoother texture and higher overall aceeptiablity. Evaluating the qualities of 12% SNF yogurt made with Lactobacillus bulgaricus CCRC 14098 (L) + Streptococcus thermophilus CCRC 12268 (S), L+S+A1, L+S+A2, L+S+B2 and L+S+E culture incubated at 27, 32, 37 and 42℃. The results showed that, the thread forming property, viscosity and syneresis of L+S+A1, L+S+A2, L+S+B2 and L+S+E sets were significantly better than L+S set (P<0.05), and 32℃ group was significantly better than others (P<0.05). Comparing the qualities of ropy yogurt and traditional yogurt containing pectin. It was found that, the thread forming property and viscosity of traditional yogurt were significantly increased with the amount of SNF and pectin increased (P<0.05), and the syneresis was significantly decreased with the amount of SNF and pectin increased (P<0.05). In addition, the thread forming property, viscosity and syneresis of ropy yogurt were still significantly better than the traditional yogurt containing 0.5% pectin (P<0.05).

碩士
南臺科技大學
生物科技系
105
Lactic acid bacteria belong one of the probiotics and maintain the intestinal health, its polysaccharide is also widely used in food additives such as thickeners, and that have the ability to enhance immunity. Our aim was to screen the exopolysaccharide-producing lactic acid bacteria and study its polysaccharide production conditions. The lactic acid bacteria strains were isolated from fermented food were screened by catalase test and Gram stain, P2 strain was selected that have high exopolysaccharide yield and ability for stimulate to macrophage. Lactic acid bacteria P2 was identified as Lactobacillus plantarum by API 50 system. In the study of polysaccharide production conditions, the best carbon sources was glucose, the yield of total exopolysaccharides and beta-(1,3)-Glucan were 376.7、302.4 mg/L, respectively; the best nitrogen sources was peptone, the yield of total exopolysaccharides and beta-(1,3)-Glucan were 320.7、297 mg/L, respectively; the optimum culture temperature of the production exopolysaccharide of L. plantarum P2 was 23℃ under no pH control to obtain the highest yield of total exopolysaccharides and beta-(1,3)-Glucan were 364 and 342.8 mg/L, respectively.

碩士
東海大學
畜產學系
90
Ropy lactic acid bacteria were used to manufacture ropy caprine milk yogurt, in order to improve the qualities of caprine milk yogurt. Four ropy strains, Streptococcus thermophilus A1, A2, Lactobacillus bulgaricus B2, E and two commercial strains, Streptococcus thermophilus CCRC12257 (S) and Lactobacillus bulgaricus CCRC14009 (L) were used. Ropy strains were incubated in 10% TS caprine milk at 32 and 42℃ for 72 hours. The pH value of samples decreased during the incubation. The bacterial counts, thread forming properties and viscosity displayed a quadratic curve. The exopolysaccharide (EPS) concentration had a trend toward reduction. The lactic acid production, maximal bacterial counts and viscosity of samples fermented at 42℃ greater than those were fermented at 32℃, while A1 and A2 sets were predominant. The thread forming properties and EPS concentration of samples fermented at 32℃ greater than those were fermented at 42℃, while A1 and A2 sets were predominant. Caprine yogurt were made at 27, 32, 37 and 42℃ by single ropy strains or mixed strains .The bacterial counts of all samples reached 108 cfu/ml. The thread forming properties and viscosity of ropy yogurt were higher than those of commercial yogurt at all incubation temperature. The incubation temperature of 32 and 37℃ resulted in the higher viscosity while the A1 and A2 sets were better than B2 and E sets. The mixed strains sets of ropy and ropy strains were better than the mixed strains sets of ropy and commercial strains. The syneresis of ropy yogurt were lower than commercial yogurt at all incubation temperature and the incubation temperature of 32 and 37℃ resulted in the lower syneresis while the A1 and A2 sets were lower than B2 and E sets. The mixed strains sets of ropy and ropy strains were lower than the mixed strains sets of ropy and commercial strains. Ropy yogurt had the least amounts of EPS at 27℃.The mixed strains resulted in less amounts of EPS than single strains, but did not affect the other characteristics. There’s no relationship between the viscosity and EPS concentration. The rheology of caprine yogurt was mainly affected by the incubation temperature. The gel strength of ropy yogurt increased with the increased incubation temperature. There’s no difference between ropy yogurt and commercial yogurt at the same incubation temperature. The ropy yogurt had the higher viscosity and the lower syneresis than commercial yogurt during the storage. The appearance and mouth-feel of ropy yogurt were higher than those of commercial yogurt. The flavor and overall acceptability of ropy yogurt were higher than those of commercial yogurt at the beginning of storage, but there’s no difference after 2 weeks.

碩士
國立臺南大學
自然科學教育學系碩士班
93
Five species of high exopolysaccharide（EPS）lactic acid bacteria were isolated from local fermented milk and identified as Lactobacillus delbrueckii subsp. bulgaricus, Leuconostoc spp., L. rhamnosus, L. delb. lactis and L. paracasei. The L. delbrueckii subsp bulgaricus had the highest EPS production assay among the five species bacteria, above all, L. paracasei was the first isolated from Taiwan milk products. The cultural studies had shown that the better microbial counts and EPS products for five species of lactic acid bacteria were under MEPS-2 medium at 15℃. Post- fermented milk culture medium at 37℃ for 24 hours followed by at 10℃ for 48 hours and single-strain starter culture instead of mixed-strain starter culture had maximum EPS production. The mean productions of EPS was 1.5 times and microbial counts was 103-106 times in single-strain starter culture compare with mixed-strain starter culture. It also showed the same trend of EPS products and microbial counts. The results suggested that the EPS production was suppressed by mixed-strain starter culture.

碩士
輔仁大學
食品營養學系
90
During July 1998 through January 1999, vegetable salads had been sampled from fast-foods chain stores in Taipei for analysis. Results indicated that Coliforms was detected in 30 samples, and the total aerobic count of 103-104 CFU/g were found in 22 out of 63 samples. E. coli and S. aureus were not found in all samples. Tenfold concentrated crude bacteriocin solutions produced from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis and Pediococcus pentosaceus inhibited Aeromonas hydrophila, Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus and Salmonella enteritidis (p<0.05). The inhibition effect of at 7℃was much better than that at 25℃. The antimicrobial activity of concentrated bacteriocin solutions was best at pH 3, however it decreased with increasing pH. Tenfold concentrated bacteriocin solution produced by P. pentosaceus had the strongest inhibition effect. It was stable at various pH values and still possessed the ability to inhibit pathogens even at pH7. Immersion of filter paper containing pathogens in tenfold concentrated crude bacteriocin solutions produced by P. pentosaceus effectively inhibited the growth of pathogens. After immersion for 60 min, it completely destroyed A. hydrophila, E. coli O157:H7, L. monocytogenes and S. aureus negative and reduced 2.6 log CFU/g of S. enteritidis. Spraying filter paper containing pathogens with tenfold concentrated bacteriocin solutions produced by P. pentosaceus showed complete inhibition of A. hydrophila, E. coli O157:H7 and S. aureus after 120 min of treatment. It also reduced 2.6 log CFU/g of L. monocytogenes. Crude bacteriocin solution without concentration significantly inhibited E. coli O157:H7 and S. enteritidis, it did not inhibit the growth of A. hydrophila, L. monocytogenes and S. aureus, even after 120 min of treatment.

碩士
弘光科技大學
食品科技所
106
Lead is common heavy metal which is toxic to humans. Widely utilized in many industries, it can contaminate the environment by being discharged into the air and water, and then enters the human body through the respiratory tract, food, or skin contact. Lead possesses the phenomena of bioaccumulation and biomagnification, and is also regarded as a persistent environmental pollutant. Lead half-life in blood is about 30 days and can remain in the skeletal system of years. Lead damages the human nervous, hematopoietic, renal, and endocrine systems. Biosorption is increasingly valued. Many biological absorbents (such a bacteria, algae, yeast and fungi) can be used to remove heavy metals. The purpose of this project is to screen strains of lactic acid bacteria (LAB) with high extracellular polysaccharide production from the strain library and evaluate the ability of LAB to absorb and eliminate heavy metals and lead (II). We further evaluate the ability of LAB to probiotic properties. The results showed the strain screened by the strains library which ability to produce extracellular polysaccharides and the amount of biomass. Used eight kinds of disks for antibiotic susceptibility tests, the results showed three strains of lactic acid bacteria were not resistant to Neomycin, Kanamycin and Streptomycin. Through API 50, PCR identification, the results from analysis were identified as Pediococcus acidilactici. The adhesion of intestinal epithelial cells assays showed the strain 17064 exhibited stronger adherence to the Caco-2 cell line. The results of the binding assays by three bacteria scavenging lead (II) ranged from 14.09 ~ 14.56 %. The lactic acid bacteria powders scavenging ability was better than bacteria. The results showed scavenging capability being 42.55 ~ 48.69 % The strains screened in this test show that the LAB have the potential to bind to metals, prevent them from entering the body and protect the host.

碩士
嘉南藥理科技大學
生物科技系暨研究所
95
Among the variety of antimicrobial substance produced by lactic acid bacteria (LAB), bacteriocins are one of the most promising natural food preservatives. Because of the inhibitory activity of bacteriocins against a variety of food spoilage-causing and/or competing organisms, bacteriocin producing LAB have been widely used in dairy, meat, and vegetable fermentations. The main purpose of this research is screening, identification and purification conditions of high productivity bacteriocin- producing Lactic Acid Bacteria Strain for bacteriocin. Lactobacillus plantarum No.13, which is a bacteriocin producer isolated from kimchi. Kinetic study of L. plantarum No.13 showed that the production of antibacterial activity started at the fourth day after inoculation, then gradually increase to reach a maxium at the 24 hours. Bacteriocin can be recovered by cells adsorption- desorption. The specific activity of the purified bacteriocin was 256.9 AU/mg, with 1.5% recovery. Bacteriocin has been described as the Class II produced by a strain of L. plantarum No.13, with a molecular mass lower than ca. 14 kDa. The bacteriocin had a high substrate specificity for lipase and was inhibited by trypsin. The bacteriocin were active against Staphylococcus aureus. Optimal pH and temperature of the purified bacteriocin from L. plantarum No.13 was stable in 2.0 and below 70℃, respectively.

碩士
國立臺灣大學
動物科學技術學研究所
106
Low-fat dairy products with reduced calories and fat might have the good market potential to meet the consumers’ need. However, removal of fat in dairy products creates several physical defects including poor texture, black color development and lack of flavor. During the process, the addition of polysaccharides could perform as stabilizers or water-binding agents to increase fermented dairy properties. Additionally, the consumers are preferring to buy “clean label” products with less artificial food additives. Exopolysaccharides (EPS) from lactic acid bacteria (LAB) are widely used in the dairy products such as yogurt, cheese, and butter. Based on their EPS-producing ability, LAB could be classified into ropy and nonropy. Ropy starter cultures could produce EPS, a natural water-binding agent, which improves moisture retention and reduces whey off in dairy products. Taiwanese ropy fermented milk (TRFM) is a dairy product showing great extension ability and viscosity. It is also referring to Taiwanese viili due to the similarity of texture and flavor to a traditional Nordic fermented milk, viili. In this study, we would like produce a fermented milk with ropy LAB, isolated from TRFM. A total of 40 isolates were selected from Taiwanese ropy fermented milk. After Gram staining test to remove Gram-negative bacteria and yeast, 34 isolates were remained. Isolates were then selected according to the flame-like suspension in MRS broth. These isolates were further typed and identified using random amplified polymorphic DNA-polymerase chain reaction PCR (RAPD-PCR), 16S rRNA sequencing and API CHL 50. Results indicated that 13 isolates were categorized into 2 different groups based on their RAPD-PCR profiles. All of them were identified as Lactococcus lactis subsp. cremoris using 16s rRNA gene analysis and API50. One strain was picked from each group and compared their EPS production. No significant difference was found between Lactococcus lactis subsp. cremoris APL15 (APL15) and Lactococcus lactis subsp. cremoris APL16 (APL16) in EPS production (P>0.05). Therefore, both strains were selected for fermented milk production. For fermented milk production, 1% APL15 and APL16 isolated from TRFM were inoculated in low fat milk at 26°C for 24 hours. Physiochemical and microbial properties were analyzed. Results indicated that both samples with APL15 and APL16 could reach pH 4.3 and acidity 0.7% in 24 hours, which is a necessary condition for a commercial fermented milk product. Viable cell counts in both samples were higher than 109 CFU/g, an important characteristic for probiotic products to provide health benefits on human beings. The results of syneresis and rheology tests also showed that no difference was found among samples and commercial fermented milk product with stabilizer, indicating EPS from LAB can act as stabilizer in products. For gastrointestinal stresses test, pH 3 and bile salt 0.5% were lethal conditions for APL15 and APL16. Stress adaptation and microencapsulation might improve survival rate under human gastrointestinal stresses. After freeze drying, the bacterial counts for both strains were higher than 108 CFU/g. This study showed that it was possible to reduce or replace commercial stabilizers in the low-fat fermented milk production with EPS producing LAB.

碩士
明志科技大學
環境與安全衛生工程系環境工程碩士班
107
Lactic acid bacteria (LAB) are one of the representative probiotics in gut, and many LAB strains have been applied widely in medical and health foods fields. These specific LAB strains are shown to could promote the intestinal health, alleviate the allergy responses or improve immune function. On the other hand, lactoferrin is a multi-functional protein and it also displays activities in antibacterial, anti-microbial, anti-inflammatory and immune regulation. Thus, in this study, some specific LAB strains were selected to express different lactoferrins to could combine the health-promoting benefits by using the two agents together. By the help of biotechnology approaches, the lactoferrin-harboring plasmids had been successfully transformed into selected LAB strains by using electroporation approach. Then, the lactoferrin-harboring LAB strains were further maintained and isolated by the help of antibiotic selection. Finally, we got 12 LAB strains (nisin-induction system) to could express lactoferrin. Then, the nisin was used to induce the expression of lactoferrin in LAB strains. The SDS-PAGE and Western blotting approach were applied to confirm the expression levels of lactoferrin in these LAB strains. Our data show that the optimal concentration of lactoferrin in LAB can be achieved by 6-hours of induction (1 ng/mL nisin). Finally, the expressed lactoferrin in LGA-LFA strain had been observed to display anti-bacterial activities. We will evaluate the expression level and functions of more obtained lactoferrin-expressed LAB strains in our next study.

碩士
國立中興大學
食品暨應用生物科技學系
95
The theme of this study is mustard pickle. We investigate mustard pickle and mustard pickle brine respectively. The study can divide into two chapter: the first chapter is “Isolation of Exopolysaccharide-Producing Probiotic Lactic Acid Bacteria from Mustard Pickle in Taiwan ” and the second chapter is “ Practicability of Making Salted Duck Eggs Using Mustard Pickle Brine ”. The first chapter: this experiment collected mustard pickle form north (Beipu Township, Hsin Chu County), midland (Tapi Township, Yunlin County) and south (Mei Nong Township, Kaohsiung County) in Taiwan to isolated exopolysaccharide (EPS)-producing lactic acid bacteria (LAB). We evaluated probiotics property and EPS production related to media of different glucose concentration and then identifies the LAB. Based on initial characterization including acid production (positive), catalase (negative), Gram-stain (positive) and cocci or rods shape, thirty nine strains were isolated from mustard pickle. By evaluating their EPS production, strain SLC 13, which was isolated from mustard pickle of Mei Nong Township Kaohsiung County, produced the maximum EPS (more than 400 mg/L) among these isolates. Through acid tolerance test, bile tolerance test, imitation of digestional tract condition test, antibacterial test and antibiotic susceptibility test, we found that LAB SLC 13 performed better than the other isolates and has the potential to be a probiotic. Further identification of LAB SLC 13 was performed by using API 50 CHL and 16S rDNA, results showed that LAB SLC 13 is Lactobacillus plantarum with more than 99 % confidence. By culturing L. plantarum SLC13 in MRS broth/ 100 ml with 6 g glucose, high EPS production (EPS 2.03 ± 0.05 g/L) and good growth (viable counts 8.88 ± 0.05 log10 CFU/mL) was observed. The finial pH and residual sugar was found to be 3.54 ± 0.01 and 0.53 ± 0.01 g/L, respectively. The experiment isolated EPS-producing and probiotics lactic acid bacteria successfully from mustard pickle in Taiwan. The second chapter: mustard pickle brine with NaCl content 18.84 ± 0.23 %, pH 4.96 ± 0.03, and total acidity level of lactic acid of 0.54 ± 0.04 %, was from the area for making mustard pickle in Tapi Township Yunlin County in Taiwan. Mustard pickle salt is made from mustard pickle brine by evaporating crystallizer. We used both mustard pickle brine and mustard pickle salt for preparing saturated brine to make salted duck eggs. Salted duck eggs making from saturated salt brine preparing from commercial salt were used as control. The changes of egg yolk and egg white during pickling time were analyzed. The weight of mustard pickled eggs was slightly decreased during pickling time; however, weight increase was observed in control group. For egg yolk, increase of the parameters such as yolk index, NaCl content, total solids and hardness were observed during pickling time with moisture content as the exception. For both experimental and control groups, decrease in parameters for egg white including albumen index, pH, G’, G’’ and η* were found. On the contrary, increase in NaCl content, total solids and tan δ were observed. In sensory evaluation using nine point hedonic scales, results showed highest scores of mustard pickled eggs in appearance, color and texture in both egg yolk and egg white were obtained. Overall acceptance also gained higher scores. Our results showed that mustard pickle brine can make salted duck eggs with mustard pickle flavor and these salted eggs can be a speciality of Tapi Township Yunlin County or anywhere that produce mustard pickles. This experiment also indicated the possibility of reusing mustard pickle brine and mustard pickle salt by developing new salted egg products.

博士
大同大學
生物工程學系(所)
96
The results are divided into two parts with screen of probiotic Lactic acid bacteria and constructing shuttle vector of Lactic acid bacteria. In part I, the probiotic Lactic acid bacteria (LAB) was screened and used for provide intestinal health and host of shuttle vector of LAB. In part II, a cryptic LAB plasmid was isolated and the plasmid sequence was determined. Based on LAB plasmid, a shuttle vector was constructed, it could maintain stably in probiotic LAB and express heterologous protein gene. In screen of probiotic LAB, the twenty-one of LAB strains which could withstand high acid and bile salt conditions were selected. These strains meanwhile could tolerate digestive system, restrain pathogenic bacteria growth, metabolize isomaltooligosaccharides and gentiooligosaccharides, and induce cytokine production (IL-12 p40p70 and IL-10) by RAW 264.7 macrophage cell. All of these strains could be used as a candidate for probiotics. The species were identified as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus by API and 16S rRNA. Strain E1, E33, E40, and E55 were plasmid free and selected as the host of shuttle vector system of LAB. In constructing shuttle vector of LAB, a cryptic plasmid pL2 was isolated from Lactococcus lactis subsp. lactis and its complete nucleotide sequence was determined (5,299-bp, GenBank accession No. DQ917780). Its replication mode was identified as a theta-type belonging to the pAMβ1 family. Analysis of the nucleotide sequence revealed that pL2 contained a transfer origin, a replication origin, and five putative open reading frames (ORF 1-5). ORF1 (386 amino acids) was homologous to replication protein RepB. The shuttle vector pUL6erm was constructed by using a replicon from pL2, a multiple cloning site, colE1 ori, the origin of Gram-negative bacteria from vector pUC19, and the erythromycin resistance gene from pVA838 as a selective marker. The pUL6erm could be transformed easily and maintained stably in E. coli, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, and Streptococcus thermophilus. The expression plasmid pUL6erm- gadR-GUS was constructed base on pUL6erm and a chloride-inducible gene expression cassette encoding gadR and the Pgad promoter. Growth in the presence of 0.3 M sodium chloride and 50 mM glutmate, the beta-D-glucuronidase was induced and expressed with 2.37 Unit/mg by plasmid pUL6erm-gadR-GUS.

碩士
國立中興大學
食品科學系
86
Nineteen strains of lactic acid bacteria including Bifidobacterium longum B6 and 15708, Lactobacillus acidophilus B, E, Farr, LA-1, N-1, and 4356, Lactobacillus bulgaricus Lb, 448, 449, 1006, 11842, and 12278, and Streptococcus thermophilus MC, 573, 821, 3641, and 19987 were investigated for cholesterol reducing ability in vitro. Most of the strains tested were able to reduce cholesterol. L. bulgaricus 449 demonstrated the best cholesterol reducing ability when oxgall was present. L. acidophilus 4356 also reduced cholesterol very well when oxgall was used. These two strains reduced cholesterol at 51 and 41 %, respectively. No particular genus or species showed higher cholesterol reducing ability than the others. The mechanisms of cholesterol reduction were also identified in the in vitro system. Although the coprecipitation of cholesterol with deconjugated biles contributed to the reduction of cholesterol levels, cholesterol was reduced when taurocholic acid was used as the source of bile. Therefore, the cholesterol reduction in the presence of bile does not attribute only to the coprecipitation with deconjugated bile salts but also to the assimilation by cells.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1994.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 184-215).

Dissertação de mestrado em Bioinformática
Biotechnology plays an essential role in the modern industry and in guaranteeing sustainable future for humankind. Advances of metabolic engineering and systems biology allow the adaption of complex cellular networks for the production or uptake of certain molecules, with great economical interest, enabling the creation of cell factories. Among the potential microorganisms that fit this role is the well-known group, due to their role in food fermentation and, in particular, their use in dairy industry, known as Lactic acid bacteria (LAB). Their metabolism is known for its relative simplicity and lack of biosynthesis capacity, creating a potential application as a cell factory in transformation processes. The purpose of this work is to develop through evolutionary engineering a strain of LAB capable of utilizing mannitol as the sole carbon source and identify mutations in the evolved strain, with the objective of associate these mutations with the mannitol consuming phenotype. Through the usage of adaptive laboratory evolution (ALE), several strains of LAB were evolved and a selected evolved strain of Lactococcus lactis subsp cremoris, capable of consuming mannitol as the sole carbon source successfully, was sequenced using next-generation sequencing. From the analysis of this genomic data using several bioinformatics tools available, 3 mutations affecting the genes pta, adhA and mtlF were identified as likely having an impact in the new phenotype presented by the evolved strain. This work provides an initial inquiry into a potential application of brown algae, which accumulate mannitol, as a new feedstock for biofuel production using LAB as cell factories.
A Biotecnologia tem assumido um papel preponderante nos processos industriais da atualidade, tendo em vista a conjugação destes com a questão da sustentabilidade da espécie humana. Os avanços na engenharia metabólica e na biologia de sistemas tem permitido a adaptação das complexas redes celulares, com o intuito de produzir ou consumir certos compostos, de forma a aumentar o seu valor económico, criando ‘fábricas celulares’. Entre os potenciais organismos para este tipo de aplicação encontra-se um grupo bastante conhecido devido à sua função na fermentação de certos alimentos, especialmente lacticínios, denominadas bactérias ácido-lácticas. Estas possuem um metabolismo relativamente simples e não apresentam várias capacidades biossintécticas, tornando-as em potenciais candidatas a serem usadas como ‘fábricas celulares’ em processos de transformação. Neste trabalho pretende-se adaptar através de engenharia evolutiva várias espécies de bactérias ácido-lácticas à utilização de manitol como fonte de carbono e proceder à identificação de mutações no genoma das estirpes evoluídas através de tecnologias de sequenciação de ADN, com o propósito de relacionar estas mutações com o fenótipo capaz de consumir manitol. Com recurso à engenharia evolutiva, várias estirpes de bactérias ácido-lácticas foram evoluídas e uma dessas estirpes, Lactococcus lactis subsp cremoris, capaz de consumir manitol como a única fonte de carbono, foi selecionada para ser sequenciada com recurso tecnologias de sequenciação de ADN. Através da análise destes dados genómicos usando várias ferramentas bioinformáticas, foi possível determinar 3 mutações que afectam os genes pta, adhA e mtlF que possivelmente estarão relacionadas com o fenótipo exibido pelas espécies evoluídas. Este trabalho serve como uma avaliação ao potencial da utilização de algas castanhas, que acumulam manitol, como um novo recurso para a produção de biocombustíveis, usando bactérias ácido-lácticas como ‘fábricas celulares’ para a sua transformação.

碩士
國立中興大學
食品科學系
92
The stimulatory effect of eight strains of lactic acid bacteria including L. acidophilus 4356, L. acidophilus LA-1, L. bulgaricus 448, L. bulgaricus 449, B. longum B6, B. longum 15708, S. thermophilus MC and S. thermophilus 573 on murine macrophage J774A.1 cell line were examined. The fractions of lactic acid bacteria including viable bacteria, heat-killed bacteria, intracellular extract, extracellular extract, crude cell wall, chromosome DNA, and whey of fermented milk were used for this study. The testing items include proliferation, the secretion of TNF-α and IL-6, and phagocytosis of murine macrophage J774A.1 cell line. The result showed that lactic acid bacteria activate the macrophages proliferation by 107 cfu/ml of viable bacteria, fermented milk and 0.05 μg/ml of genomic DNA, respectively. And the degree depended on the difference of strains and numbers of bacterium. Co-culture with 1,000 and 100 μg/ml crude cell wall extract or whey of fermented milk which containing 106-108 cfu/ml lactic acid bacteria with macrophage cell line significantly increased its production of TNF-α. Crude cell wall extracts (1,000 and 100 μg/ml) and viable bacteria (107 cfu/ml L. acidophilus LA-1, 107-108 cfu/ml, L. bulgaricus 448, 106-107 cfu/ml B. longum 15708 and S. thermophilus MC) could stimulate the production of IL-6 by macrophage cell line. Many fractions of lactic acid bacteria could stimulate phagocytosis of macrophage cell line, including heat-killed bacteria, intracellular extract, extracellular extract, genomic DNA and whey of fermented milk. Immunomodulatory activity of lactic acid bacteria depended on difference of strains and numbers of bacterium, and expressed strain-dependent manners. In our results also indicate that lactic acid bacteria can stimulate the macrophage cell line and may improve immune system, but not to such an extent as to inflammation.

碩士
中興大學
食品暨應用生物科技學系
95
Abstract The key technique of bread production is the improvement of flavor and storage time and the addition of lactic acid bacteria (LAB) to bread during the manufacturing process would help. In this study first screened LAB with the ability to leaven the sourdough and produce good flavor. Among the 16 tested strains, Lactobacillus delbrueckii delbrueckii (LDD) produced the most acidity and Pediococcus acidilactici (PA) produced the best flavor. We then used these two strains in toast making. LDD and PA were added into sourdough which was made to toast, and the proximate composition, physical parameters, taste quality, volatile components, bioactive components and sensory quality of toast were analyzed. Experiment results indicated that the lightness of the three toasts were in the descending order of LDD toast（71.20）>white toast（69.97）> PA toast（68.98）. X-ray diffraction revealed that the peak strength of the three toasts increased during the storage time. In a six-day storage experiment at 25℃, the hardness of the three toasts increased, while hardness, cohesiveness, gumminess, chewiness and resilience decreased with time. The LDD toast was the most abundant in nucleotides content (1.95 μg/g), following by white toast (1.11 μg/g) and PA toast (0.97 μg/g). Also, the LDD toast contained the most organic acid (4.07 mg/g) and PA toast (4.35 mg/g) following by white toast (3.10 mg/g). LDD toast contained the most kinds and quantity of aromas. In the six-day storage experiment, the total plate count of PA toast (3.20 log CFU/g) and LDD toast (46 log CFU/g) were significantly lower than white toast (7.85 log CFU/g) (p＜0.05). With regard to the hedonic scores in sensory evaluation, LDD toast and PA toast were more accepted than white toast in flavor and mouth feel. In a summary, the addition of LDD to toast enhanced the palatable taste and sweet taste, and thus improved the flavor and nutritional value of toast.

碩士
中國醫藥大學
藥學系碩士班
97
Lactic acid bacteria isolated from soil, plants, fermented dairy products, animal feces or human feces are considered to be probiotics with therapeutic and healthy properties; however, the effectiveness of probiotics can be affected by manufacturing processes, such as freeze-drying or spray-drying. The purpose of this study was to develop a technique for microencapsulation of probiotics that can protect probiotics during the freeze-drying process. The formulations(A-MsP) of alginate (3%) with 3% skim milk powder, and 2% peptone were prepared. The effect of the cryoprotectants on cell survival during freeze-drying was investigated using a standardized amount of cells and the optimized freeze drying media. The results showed that this formulation (A-MsP) provided sufficient protection for probiotics, resulting in probiotic counts of up to 1011 CFU/g after freeze-drying. The survival of microencapsulated L. rhamnosus was better than free cell at low pH. The release of encapsulated cells in simulated aqueous solution of colonic pH was also assessed. However, this formulation (A-MsP) needs to be improved if we want to apply microencapsulation to spray-dry. The microencapsulated lactic acid bacteria maintain their survival at 4℃ during a 6 week period. This probiotic microencapsule can potentially be used in fermented dairy products and healthy food products or as a drug delivery system in the future.

碩士
國立臺灣海洋大學
食品科學系
102
gamma-Aminobutyric acid (GABA) is a well-known non-protein amino acid, which is widely distributed in animals and plants. GABA has been reported to be a major inhibitory neurotransmitter in the mammalian central nervous system and show antihypertensive activity. This aims of this research are to manufacture a GABA-enriched fermented milk by lactic acid bacteria (LAB) isolated from fish intestines and to evaluate the probiotic potential property of the GABA-producing LAB. Preliminarily, lactic acid bacteria strains were isolated from healthy fish intestine from previous study. Ten lactic acid bacteria strains of 126 LAB strains were screened based on the capacity of synthesizing GABA. And ten GABA-producing strains show that none of them exhibited hemolytic activity. Moreover, five strains exhibited partial bile salt hydrolase activity, including the strain FPS 2520. FPS 2520 also showed a high percentage of adhesion to monolayer of Caco-2 cells. Strain FPS 2520 demonstrated high survival viability to gastrointestinal conditions simulating stomach and duodenum passage. Optimal conditions of strain FPS 2520 for producing GABA in whole milk were: 10% reconstituted whole milk, initial inoculum size of 5 log CFU/mL, addition of 20 mM MSG, 1% brown sugar, and 0.5% yeast extract during fermentation at 37oC for 48 hours. The results indicated FPS 2520 show a probiotic potential property and have a great application in milk fermentation for the production of G (10.67 mg/mL), and the IC50 of ACEI-inhibitory activity is 0.28 ± 0.02 mg/mL. Besides lactic acid bacteria number slightly decreased, pH value, titratable acidity and GABA concentration didn’t have significant difference compare to control at 4oC for 14 days. Using mixed strains, GABA-producing strain FPS 2520 and proteinase-positive FKR 3737 could significantly enhance the GABA production about 14.74 mg/mL.

博士
國立中興大學
食品科學系
91
Banana was used as the raw material for the preparation of fermentation media of Lactobacillus acidophilus, and cell immobilization was applied to improve the fermentation efficiency of L. acidophilus in banana media. Cell immobilization was performed using calcium alginate and κ-carrageenan as the entrapping matrix, and gel beads of diameters around 2.6 mm for the former and 3.0 mm for the latter were obtained. Both green and ripe bananas were used for the preparation of banana media, and both free and immobilized cells were used to conduct the fermentation for 80 hours. The viable cell number in ripe banana media was found to be higher than that in green ones during both free cell and immobilized cell fermentation. During the fermentation of immobilized cell, cells would leak out from the gel beads and grew in the medium solution. In immobilized cell fermentation, the final viable cell number could reach 105 CFU/ml in medium suspension and that in gel beads could become over 108 CFU/ml gel. In free cell fermentation, the final viable cell number was around 106 CFU/ml. Immobilized cell could overcome the unfavorable conditions in green banana media and improved results could be obtained. During the fermentation, the variation of pH and titratable acidity showed obvious relationships with the growth of cells. Variation of fructooligosaccharides contents in ripe banana media was not remarkable in immobilized cell fermentation compared to free cell. Immobilized L. acidophilus fermented banana medium was able to be used as a synbiotic product by combining the probiotic effect of L. acidophilus and the prebiotic effect of banana. The effect of Ca-alginate immobilization was better than κ-carrageenan. Based on the overall results of cost analysis, Ca-alginate immobilization was a better choice compared toκ-carrageenan immobilization. On the other hand, L. acidophilus was immobilized using Ca-alginate andκ-carrageenan, and protection effects of cell immobilization on the viability of the bacteria after freezing and freeze-drying were studied, and its influence on the storage stability of the freeze-dried cells at 5℃, 25℃, 45℃, 60℃, 70℃ was also investigated. Initial concentration of both free and immobilized cells used for experiments all reached the level of 1010 cells/ml. Results indicated that the immobilization in Ca-alginate gel beads andκ-carrageenan gel beads could provide effective protection to reduce the damage of bacteria under operations. High correlations were obtained between Log D values and storage temperatures for both free and immobilized cells under those various storage temperatures used. the z value which derived from the linear regression equation of Log D and storage temperature for free and immobilized cells were significantly different (p < 0.05). Cell immobilization could enhance temperature tolerance of the freeze-dried bacteria during storage and diminish the influence of temperature variation on the storage stability of freeze-dried cells.

碩士
中華醫事科技大學
生物醫學研究所
100
Diabetes has become a global disease, which is accompanied by complex complications, reinforce the seriousness of diabetes, so in terms of treatment and prevention, has become urgent and important research topic. Health Department announces 2010 citizens 10 leading causes of death, diabetes has been highest in the fifth of the ten major causes of death, and the proportion of men and women. Diabetes is a will effect multiple organ of metabolism sexual disease, and is high medical spent of disease, long-term complications including retinal lesions and has potential occurs blind of dangerous, and kidney lesions caused kidney function failure, and perimeter neuropathy variable and has foot Department ulcer and amputation of dangerous and the neural sexual joint disease, and independent neuropathy variable caused stomach, and reproductive urinary and cardiovascular symptoms and the sexual function obstacles,, are will on patients and family caused psychological and social function of impact. Currently on the clinical treatment of diabetes complications are mainly controlled condition deteriorated and reduce complications, however traditional drug therapy is often limited, and is prone to side effects, so diet is the most natural method, it aims to make blood sugar close to normal values, and the maintenance of blood fat and cholesterol of normal, and achieve the goal of normal weight. In the past, many studies pointed out that the lactic acid bacteria to decrease blood lipids, lowering blood sugar, lowering blood pressure, and activation of the immune system and other effects. Therefore, if the use of lactic acid bacteria supplements, believe, will help relieve or prevent the symptoms of some of the complications of diabetes and incidence for complications of diabetes, kidney disease, cardiovascular and cerebral vascular disease, dental complications, this study and discussion, that the effectiveness of lactic acid bacteria, their symptoms improve or prevent.