Boza kaynaklı laktik asit bakterilerinin laktik asit miktarlarının ve staphylococcus aureus üzerine antimikrobiyal etkinin belirlenmesi
Determination of the amount of lactic acid obtained from boza-based lactic acid bacteria and its inhibation effect aganist staphylococcus aureus
Yazar: GÖZDE GÜLSEREN
Danışman: PROF. DR. Z.DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Lactobacillus = Lactobacillus ; Lactococcus = Lactococcus

Özet:
Laktik asit bakterileri, değişik metabolik aktiviteleri sayesinde özellikle fermente gıda sanayisinde önemli rol oynamaktadır. Bakteriler aynı zamanda starter kültür olarak, gıda katkı maddesi olarak ve diğer üretmiş olduğu metabolitler dolayısı ile de fermentasyon ve diğer işlevler için yardımcı unsur olarak kullanılmaktadır. Ayrıca, laktik asit bakterileri peynir gibi süt ürünlerinin oluşumunda ve olgunlaşmasının hızlanmasında, gıdaların besinsel, teknolojik ve organoleptik özelliklerin geliştirilmesinde rol oynamaktadır. Laktik asit bakterilerinin, laktik asit, bakteriyosin ve diğer antimikrobiyal özellik gösteren maddeler dolayısı ile gıdaların doğal yollarla korunması ve özellikle patojen mikroorganizmaların ve diğer istenmeyen mikroorganizmaların inhibisyonu sağlanmaktadır.Staphylococcus aureus gıda sanayisinde en önemli patojen bakterilerinden biri olup, bakterinin inhibe edilmesi için önem arz etmektedir. Patojenin gıda bozulmalarına yol açarak, gıda zehirlenmeleriyle insane sağlığını tehdit etmektedir .Özellikle son günlerde kimyasal maddelerin ve katkı maddelerinin kullanımı hakkında halkın bilinçlenmesi, gıdaları da doğal yollarla korumanın önemini arttırmıştır. Bu yüzde, laktik asit bakterilerinin sentezlemiş olduğu laktik asit'in gıda korumasındaki etkisi daha da incelenmeye değer bulunmuştur.Yapılan tez çalışmasında boza kaynaklı Laktik Asit Bakterileri gruplarından olan Lactobacillus, Lactococcus ve Leuconostoc bakterinin üretmiş olduğu laktik asit izomerlerinin cins ve miktarları belirlenmiş ve laktik asit'in gıda sektöründe büyük sorunlara yol açan Staphylococcus aureus bakterisine karşı olan etki mekanizmasının incelenmesi amaçlanmıştır. Laktik asit bakterilerinin bakterinin üretmiş olduğu laktik asit izomerlerinin tayini kitler aracılığı ile yapılmıştır. Ayrıca, bu gruptaki bakterinin üretmiş olduğu laktik asit'in Staphylococcus aureus üzerindeki inhibisyon etkisi spektrofotometre kullanılarak incelenmiştir.Deney sonuçlarına gore, Lactobacillus türü bakterileri arasından, B23 Lactobacillus paracasei bakterisinin en yüksek oranda laktik asitin D(-) ve L(+) izomerilerini sentezlediği sonucuna ulaşılmıştır. Lactoccoccus türünün çalışılan iki farklı suşunun L(+)-laktik asit izomerini benzer miktarlarda sentezlediği saptanmıştır. Leuconostoc bakterilerinden en yüksek oranda D (-)-laktik asit sentezleyen bakteri suşunun ise E55 Leuconostoc lactis suşu olduğu belirlenmiştir. Ayrıca, Lactobacillus türünün ilgili suşlarından B10 Lactobacillus fermentum bakterisinin Staphylococcus aureus üzerindeki en yüksek inhibisyon değerine sahip olduğu bulunmuştur. D41 Lactobacillus plantarum bakteri suşunun ise Staphylococcus aureus üzerindeki en düşük inhibisyon değerine sahip olduğu gözlenmiştir. Lactococcus türü bakterilerinden olan F45 Lactococcus lactis bakterisinin Staphylococcus aureus üzerindeki en yüksek inhibisyon değerine sahip olduğu belirlenmiştir. Ayrıca, Leuconostoc türü suşları arasında, Staphylococcus aureus üzerindeki en yüksek inhibisyon değerine A40 Leuconostoc lactis bakterisi, en düşük inhibisyon değerine ise A31 Leuconostoc mesenteroides bakterisinin ulaştığı gözlenmiştir.

Abstract:
Lactic acid bacteria (LAB) are gram-positive and non-spoerforming bacteria. Their shape can be cocci, coccibacilli or rods. When their DNA was monitored, it is found that their DNA base composition of less than 53mol% G+C. In addition, most of the lactic acid bacteria are non respiratory and lack catalase enzyme that is why they possess superoxide dismutase and have alternative means to detoxify peroxide radicals.Generally, all lactic acid bacteria can grow in the medium where is any oxygen but the bacteria can grow in the presence of oxygen as aerotolerant anaerobes, unlike most anaerobes.Lactic acid bacteria use the sugar, especially glucose. They convert the sugar primarily to lactic acid and other products named CO2 and ethanol. Bacteria obtain their all energy from sugar and that is why growing lactic acid bacteria can be restricted to environments in which sugars are present or not.Lactic acid bacteria refers to a large group of beneficial bacteria that have similar properties and all produce lactic acid as an end product of the fermentation process Even though many genera of bacteria produce lactic acid ferment sugar and produce lactic acid as a primary or secondary end-product of fermentation, the term ?Lactic Acid Bacteria? is conventionally reserved for genera in the order Lactobacillus, Pediococcus, Lactococcus, Leuconostoc, Strepreptococcus, as well as Carbobacterium, Enterecoccus, Oenococcus, Tetragenococcus, Vagococcus and Weisella.Lactic acid bacteria are widespread in nature and are also found in human digestive systems and they take place in the food industry. They are used for pickling of vegetables, baking, winemaking, curing fish, meats and sausages but their main role is that preparation of fermented dairy product. Lactic acid also gives fermented milks their slightly tart taste. Additional characteristic flavours and aromas are often the result of other products of lactic acid bacteria. Likewise, they are also responsible for formation and maturation of cheeses and improvement of nutritional, technological and organoleptic value of food products. In addition, the bacteria can be are used as starter cultures, as additives and as helping metarials that are produced by them for fermentation or other process.They have an significant role in also and cereal based product process. For example, especially, the cereal based fermented products are used in all over the world as raw materials. That is why, they take place in big global food production. ?Boza? is the one of the most important fermented beverages and it is our one of the traditional fermented beverage which is consumed by Turkish people very much. Boza is produced using boiling cereals that are millet, rice, corn, millet or wheat and then saccharose sugar is added and the fermentation process is began. In this study, ?Boza? based lactic acid bacteria were used to carry out to obtain lactic acid for the assays.In addition, lactic acid bacteria have long been used to preserve and keep the nutritive qualities of various food products. LAB produce various antimicrobial compounds, which can be fall into two groups. First group can be named as low-molecular-mass compounds which are hydrogen peroxide, carbon dioxide, diacetyl and uncharacterized compounds. Second group can be said as high-molecular-mass compounds such as bacteriocins. However, it is very clear that the primary antimicrobial e ? ect exerted by LAB is the production of lactic acid and reduction of pH. Levels and types of organic acids produced during the fermentation process depend on LAB species or strains, culture composition and growth conditions The antimicrobial e ? ect of organic acids lies in the reduction of pH, as well as the undissociated form of the molecules. All of these antimicrobial substances can antagonize the growth of some spoilage and pathogenic bacteria in foods. Lactic acid bacteria have also the antagonistic property that is attributed to the lowered pH, the undissociated acids and production of other primary and secondary metabolites produced by LAB. The metabolites which are produced by the fermentation process, except the volatile ones, are kept in the foods and they leads to growth inhibition of food spoilage or poisoning bacteria and detoxification of noxious compounds of plant origin.Staphylococcus aureus is one of most important pathogen bacteria for food industry and it causes food poisoning or food spoilage in some cases. of this bacteria is important. that is why it is a threat on human health. Staphylococcus aureus is one of most important pathogen bacteria for food industry and inhibition of this bacteria is important. Staphylococcus aureus is gram-positive, non-motile, catalase and coagulase positive bacteria. It?s shape is cocci and cells can be spherical single or paired cocci or form grape-like clusters. It does not have any spore forming. Staphylococcus aureus is able to grow in a wide range of temperatures at at 7°C to 48.5°C with an optimum of 30°C to 37°C and pH at 4.2 to 9.3, with an optimum of 7 to 7.5 as well as and sodium chloride concentrations that is up to 15% NaCl. These characteristics of S. aureus preapare to grow this bacteria on variety of foods. In addition, some S. aureus strains are able to produce staphylococcal enterotoxins (SEs) and it cause to staphylococcal food poisonings. It is known that food poisonings based on enterotoxins are big threats on human health. That is why S. aureus contaminations can be readily avoided by heat treatment of food. However, they remain a major cause of food-borne diseaes because they can contaminate food products during preparation and processing. In these cases, owing to the public awareness about using chemical substances and additives food protection, the interest of food production with natural ways increases. It is observed that people want to protect your foodstuffts in natural ways and they also want to consume foods which are protected naturally. Thus, the effect of lactic acid which are produced by lactic acid bacteria on food preservation is focused more.In this study, the assays carried out two aims. First, the amounts and kind of lactic acid which are produced by Lactobacillus, Lactococcus ve Leuconostoc lactic acid bacteria group were determinated. Secondly, the inhibation mechanism of lactic acid which is also taken from Lactobacillus, Lactococcus and Leuconostoc lactic acid bacteria group against Staphylococcus aureus that leads to big problems for food industry were studied.Lactic acid have two isomers which are named D(-)-lactic acid and L (+)-lactic acid. Isomers are the molecules that have the same molecular formula but they have a different arrangement of the atoms in space. Because of whole molecule rotating or particular bonds of molecule rotating, more different arrangements occur. This phenomena is known as structural isomerism. Lactic acid isomers which are produced by lactic acid bacteria were measured using kits. In the assays, number of lactic acid bacteria were studied to obtain lactic acid taken from lactic acid bacteria which are Lactobacillus, Lactococcus and Leuconostoc bacteria species. First, Lactobacillus bacteria groups which are B2 Lactobacillus fermentum, C3 Lactobacillus fermentum, B23 Lactobacillus paracasei, D29 Lactobacillus para.sub.paracasei, E8 Lactobacillus para.sub.paracasei, F30 Lactobacillus plantarum, E32 Lactobacillus brevis, C19 Lactobacillus carpophilus, A15 Lactobacillus delb.sub.delb, C55 Lactobacillus salivarius were studied to obtain lactic acid. In addition, Lactococcus groups that are F39 Lactococcus raffinolactis and B49 Lactococcus raffinolactis were researched to take lactic acid, secondly. Finally, Leuconostoc bacteria were also investigated to obtain lactic acid which include E55 Leuconostoc lactis, B56 Leuconostoc citreum and A31 Leuconostoc mesenteroides. To determinate concentration and kind of lactic acid that are of L (+)-lactic acid and D(-)-lactic acid, D/L lactic acid kits were used. Kits had seven different solutions. In addition, the principal of kits dependes on the UV measurement and measuring the absorbance of substances. Measuring absorbances of lactic acid were carried out by means of spectrophotometer at 340 nm. Measurements in the assays were carried out duplicate.To determinate inhibition effect of lactic acid against Staphylococcus aureus, number of lactic acid bacteria which are Lactobacillus, Lactococcus and Leuconostoc species were studied. Firstly, lactic acid which is obtained from Lactobacillus bacteria groups including D41 Lactobacillus plantarum, F30 Lactobacillus plantarum, B10 Lactobacillus fermentum, E32 Lactobacillus brevis, B23 Lactobacillus paracasei, E8 Lactobacillus paracasei spp. paracasei, C19 Lactobacillus carpophilus and C55 Lactobacillus salivarius were studied. Secondly, lactic acid that is taken from Lactococcus groups which are F29 Lactococcus raffinolactis, B49 Lactococcus raffinolactis, F39 Lactococcus raffinolactis, E57 Lactococcus lactis, F45 Lactococcus lactis and A47 Lactococcus lactis spp. lactis were investigated. Finally, inhibition effect of lactic acid that are gained from Leuconostoc spices E55 Leuconostoc lactis, A40 Leuconostoc lactis, A31 Leuconostoc mesenteroides and B56 Leuconostoc citreum were studied. In addition, the inhibition effect of lactic acid against Staphylococcus aureus is measured via spectrophotometer. After obtained lactic acid from different lactic acid bacteria species, lactic acid, Staphylococcus aureus that is determinated its concentration and Mueller Hinton Broth were added into ELISA microplates at they were measured at OD660 and the measurements were taken three hours intervals until 24th hour.According to the assays, it is obvious that B23 Lactobacillus paracasei bacteria produces the highest value of lactic acid isomers which are D(-) and L(+)-lactic acid among all tested Lactobacillus species. For studied only two different Lactoccoccus strain, it is observed that they produce the same amount of L(+)-lactic acid isomers. It is clear that E55 Leuconostoc lactis strain produces the highest value of D(-)-lactic acid isomers among all tested Leuconostoc species.In addition, It is obvious that B10 Lactobacillus fermentum produced the highest value of lactic acid against Staphylococcus aureus pathogen bacteria whereas D41 Lactobacillus plantarum produced the lowest value of lactic acid against Staphylococcus aureus pathogen bacteria among tested all Lactobacillus species. It is observed that F45 Lactococcus lactis produced the highest value of lactic acid against Staphylococcus aureus pathogen bacteria among tested all Lactococcus species. Furthermore, it is clear that A40 Leuconostoc lactis produced the highest value of lactic acid against Staphylococcus aureus pathogen bacteria whereas A31 Leuconostoc mesenteroides produced the lowest value of lactic acid against Staphylococcus aureus pathogen bacteria among tested all Lactobacillus species.According to the this study facts, It can be said that lactic acid obtained from lactic acid bacteria which are studied in the assays have inhibition effect against Staphylococcus aureus. Thus, lactic acid can be used to inhibite Staphylococcus aureus bacteria.