Date of Award
5-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Food Science and Technology
Department
Food Science
First Advisor
Dr. Mutamed Ayyash
Second Advisor
Prof. Afaf Kamal Eldin
Abstract
Exopolysaccharides (EPS), produced by Lactic Acid Bacteria (LAB), are carbohydrate polymers with unique physicochemical properties and various biological activities widely used in food and pharmaceutical industries. This dissertation is aimed to (1) screen 42 new isolates from traditional yogurt-like products (Labneh) for their probiotic nature and identify the LAB strains using 16S rDNA sequencing; (2) optimize the conditions like pH, temperature, time, and quantity of sucrose for the growth of EPS produced by four selected LAB isolates; (3) purify and characterize EPS for the contents and investigate the bioactivities of the partially purified EPS; (4) study the effects of the EPS on the gut microbiome and the rheological properties of fermented skimmed bovine milk. The results showed that 10 out of 42 isolates had good probiotic potential and were identified using 16S rDNA sequencing. The LAB isolates had reductions of 4.5 to 8.5 Log10 CFU/ml after in vitro digestion. The LAB exhibited cholesterol-lowering (> 30%), pathogen-inhibiting properties, and hydrophobicity values of 7–86%, 18–87%, and 21–87% (for xylene, octane, and hexadecane, respectively). Resistance to lysozyme activity was also high in the selected LAB isolates. Out of 10, four LAB isolates Lactobacillus delbrueckii, Lacticaseibacillus rhamnosus, Enterococcus faecium, and Streptococcus thermophilus were used to produce EPS-LB3, EPS-MLB3, EPS-LB13 and EPS-MLB10, respectively. The optimum conditions to produce EPS were pH 6, at 43ºC for 48 h with 20% sucrose, and the phenol-sulfuric acid method revealed that the average EPS production was 261.4 ± 15.2 mg/L. The refractive index detector showed that the average molecular weight ranged from 1272.2 kDa to 3762.4 kDa, and the diode array detector revealed the presence of galactose, lactose, glucose, mannose, xylose, ribose, arabinose, and galacturonic acid in different concentrations when derivatized using Phenyl-3-Methyl-5-Pyrazolone (PMP). The scavenging rate of the partially purified EPS at 250 mg/L against 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), 2,2’-Azino-bis (3-ethylbenzene-thiazoline-6-sulphonic acid) radical (ABTS), superoxide dismutase, superoxide anion radical, hydrogen peroxide, hydroxyl radical, metal chelating activity, and inhibition of lipid peroxidation ranged from 10% to 88%. The antioxidant capacity for Ferric Reducing Antioxidant Power (FRAP), Reducing Power (RP), and Total Antioxidant Capacity (TAC) ranged between 714 to 2848 μg/mL, equivalent to ascorbic acid activity. The partially purified EPS inhibited the growth and biofilm formation of E. coli, S. typhimurium, S. aureus, and L. monocytogenes with an average inhibition of 53% to 74%. The average inhibition of amylase, glucosidase, cholesterol, and Angiotensin-Converting Enzyme (ACE) was 2% to 66%, and inhibition of Caco-2 and MCF-7 cancer cells was 9% to 83%. All the EPSs supported the growth of beneficial gut bacteria from Proteobacteria, Bacteroidetes, Firmicutes, and Acinetobacter in fecal fermentation with total Short Chain Fatty Acids (SCFA) production from 4530 to 6104 PPM. This study showed that the extracted EPS from the selected LABs has good bioactivities, prebiotic nature, and impact gut microbiota. Moreover, the EPS-producing bacteria improve the gelation time of fermented skimmed bovine milk. Further studies are required to investigate the EPS effect in vivo conditions and to use industrial waste for EPS production using the LAB.
Arabic Abstract
ﻋﺪﯾﺪات اﻟﺴﻜﺎرﯾﺪ اﻟﺨﺎرﺟﯿﱠﺔ ﻟﺒﻜﺘﯿﺮﯾﺎ ﺣﻤﺾ اﻟﻼﻛﺘﯿﻚ اﻟﻤﻌﺰوﻟﺔ ﻣﻦ اﻟﻠﺒﻨﺔ: ﺗﻮﺻﯿﻔﮭﺎ، أﻧﺸﻄﺘﮭﺎ اﻟﺤﯿﻮﯾﺔ، وﺗﺄﺛﯿﺮھﺎ ﻋﻠﻰ ﻣﯿﻜﺮوﺑﯿﻮم اﻷﻣﻌﺎء ورﯾﻮﻟﻮﺟﯿﺎ اﻟﺤﻠﯿﺐ اﻟﻤُﺨﻤﺮ
ﻋﺪﯾﺪات اﻟﺴﻜﺎرﯾﺪ اﻟﺨﺎرﺟﯿﺔ (EPS)، اﻟﻤُﻨﺘﺠﺔ ﺑﻮاﺳﻄﺔ ﺑﻜﺘﯿﺮﯾﺎ ﺣﻤﺾ اﻟﻼﻛﺘﯿﻚ (LAB)، ھﻲ ﺑﻮﻟﯿﻤﺮات ﻛﺮﺑﻮھﯿﺪراﺗﯿﺔ ذات ﺧﺼﺎﺋﺺ ﻓﯿﺰوﻛﯿﻤﯿﺎﺋﯿﺔ ﻓﺮﯾﺪة ﻛﻤﺎ أﻧﮭﺎ ﺗﻤﺘﻠﻚ أﻧﺸﻄﺔ ﺑﯿﻮﻟﻮﺟﯿﺔ ﻣﺨﺘﻠﻔﺔ ﺗُﺴﺘﺨﺪم ﻋﻠﻰ ﻧﻄﺎق واﺳﻊ ﻓﻲ اﻟﺼﻨﺎﻋﺎت اﻟﻐﺬاﺋﯿﺔ واﻟﺼﯿﺪﻻﻧﯿﺔ. ﺗﮭﺪف ھﺬه اﻷطﺮوﺣﺔ إﻟﻰ (١) فحص ٤٢ ﻋﺰﻟﺔ ﺟﺪﯾﺪة ﻣﻦ اﻟﻤﻨﺘﺠﺎت اﻟﺘﻘﻠﯿﺪﯾﺔ اﻟﺸﺒﯿﮭﺔ ﺑﺎﻟﺰﺑﺎدي (اﻟﻠﺒﻨﺔ) ﻟﻤﻌﺮﻓﺔ طﺒﯿﻌﺘﮭﺎ اﻟﺤﯿﻮﯾﺔ وﺗﺤﺪﯾﺪ ﺳﻼﻻت ﺑﻜﺘﯿﺮﯾﺎ ﺣﻤﺾ اﻟﻼﻛﺘﯿﻚ ﺑﺎﺳﺘﺨﺪام ﺗﺴﻠﺴﻞ rDNA16S؛ (٢) ﺗﺨﺼﯿﺺ اﻟﻈﺮوف اﻷﻣﺜﻞ ﻛﺎﻟﺮﻗﻢ اﻟﮭﯿﺪروﺟﯿﻨﻲ، درﺟﺔ اﻟﺤﺮارة، اﻟﻮﻗﺖ، وﻛﻤﯿﺔ اﻟﺴﻜﺮوز اﻟﻼزﻣﯿﻦ ﻟﻨﻤﻮ EPS اﻟﻤُﻨﺘَﺞ ﺑﻮاﺳﻄﺔ أرﺑﻊ ﻋﺰﻻت LAB ﻣُﺨﺘﺎرة؛ (٣) ﺗﻨﻘﯿﺔ وﺗﻮﺻﯿﻒ EPS وﻓﺤﺺ اﻷﻧﺸﻄﺔ اﻟﺤﯿﻮﯾﺔ لـ EPSاﻟﻤُﻨﻘﻰ ﺟﺰﺋﯿًﺎ؛ (٤) دراﺳﺔ آﺛﺎر EPS ﻋﻠﻰ ﻣﯿﻜﺮوﺑﯿﻮم اﻷﻣﻌﺎء واﻟﺨﺼﺎﺋﺺ اﻟﺮﯾﻮﻟﻮﺟﯿﺔ ﻟﻤُﺨﻤﱠﺮ اﻟﺤﻠﯿﺐ اﻟﺒﻘﺮي ﻣﻨﺰوع اﻟﺪﺳﻢ. أظﮭﺮت اﻟﻨﺘﺎﺋﺞ أن ١٠ ﻣﻦ أﺻﻞ ٤٢ ﻋﺰﻟﺔ ﻟﺪﯾﮭﺎ إﻣﻜﺎﻧﺎت ﺟﯿﺪة ﻟﺨﺼﺎﺋﺺ اﻟﺒﺮوﺑﯿﻮﺗﯿﻚ وﻗﺪ ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ اﻟﻌﺰﻻت ﺑﺎﺳﺘﺨﺪام ﺗﺴﻠﺴﻞ rDNA16S. اﻧﺨﻔﻀﺖ ﻗﺎﺑﻠﯿﺔ ﺑﻘﺎء اﻟﻌﺰﻻت ﻣﻦ ٤.٥ إلى ٨.٥ Log10 CFU/ml ﺑﻌﺪ اﻟﮭﻀﻢ ﻣﺨﺒﺮﯾًﺎ. ﻛﻤﺎ أظﮭﺮتْ اﻟﻌﺰﻻت ﻗﺪرﺗﮭﺎ ﻋﻠﻰ ﺧﻔﺾ اﻟﻜﻮﻟﯿﺴﺘﺮول (>٣٠٪)، واﻣﺘﻠﻜﺖْ اﻟﻘﺪرة ﻋﻠﻰ ﺗﺜﺒﯿﻂ ﻣُﺴﺒﺒﺎت اﻷﻣﺮاض، وأﺑﺪتْ ﺧﺼﺎﺋﺺ ﻛﺎرھﺔ ﻟﻠﻤﺎء ﺑﻨﺴﺒﺔ ٧-٨٦٪، ١٨-٨٧٪، ٦١-٨٧٪ (ﻟﻠﺰﯾﻠﯿﻦ واﻷوﻛﺘﺎن واﻟﮭﯿﻜﺴﺎدﯾﻜﺎن، ﻋﻠﻰ اﻟﺘﻮاﻟﻲ). ﺑﯿﻨﻤﺎ ﻛﺎﻧﺖ ﻣﻘﺎوﻣﺔ ﻧﺸﺎط اﻟﻠﯿﺰوزﯾﻢ ﻣﺮﺗﻔﻌﺔ ﻓﻲ اﻟﻌﺰﻻت اﻟﻤُﺨﺘﺎرة. ﻣﻦ أﺻﻞ ١٠، ﺗﻢ اﺳﺘﺨﺪام أرﺑﻊ ﻋﺰﻻت وھﻲ Lactobacillus delbrueckii و Lactobacillus rhamnosus و Enterococcus faecium و Streptococcus thermophilus ﻹﻧﺘﺎج EPS-LB3 و EPS-MLB3و EPS LB13 و EPS-MLB10ﻋﻠﻰ اﻟﺘﻮاﻟﻲ. ﻛﺎﻧﺖ اﻟﻈﺮوف اﻟﻤُﺜﻠﻰ ﻹﻧﺘﺎج EPS ھﻲ اﻟﺮﻗﻢ اﻟﮭﯿﺪروﺟﯿﻨﻲ 6، ﻋﻨﺪ °س٤٣ ﻟﻤﺪة ٤٨ ﺳﺎﻋﺔ ﻣﻊ ٢٠٪ ﺳﻜﺮوز، وﻛﺸﻔﺖْ طﺮﯾﻘﺔ ﺣﻤﺾ اﻟﻔﯿﻨﻮل-ﻛﺒﺮﯾﺘﯿﻚ أن ﻣﺘﻮﺳﻂ إﻧﺘﺎج EPS ﻛﺎن ٢٦١.٤ ± ١٥.٢ ﻣﻠﻐﻢ / ﻟﺘﺮ. أظﮭﺮَ ﻛﺎﺷﻒ ﻣﻌﺎﻣﻞ اﻻﻧﻜﺴﺎر أن ﻣﺘﻮﺳﻂ اﻟﻮزن اﻟﺠﺰﯾﺌﻲ ﯾﺘﺮاوح ﻣﻦ ١٢٧٢.٢ kDa إﻟﻰ ٣٧٦٢.٤ kDa، ﻛﻤﺎ ﻛﺸﻒَ ﻛﺎﺷﻒ ﻣﺼﻔﻮﻓﺔ اﻟﺪاﯾﻮد ﻋﻦ وﺟﻮد اﻟﺠﺎﻻﻛﺘﻮز، اﻟﻼﻛﺘﻮز، اﻟﺠﻠﻮﻛﻮز، اﻟﻤﺎﻧﻮز، اﻟﺰﯾﻠﻮز، اﻟﺮﯾﺒﻮز، اﻷراﺑﯿﻨﻮز، وﺣﻤﺾ اﻟﺠﺎﻻﻛﺘﻮروﻧﯿﻚ ﺑﺘﺮاﻛﯿﺰ ﻣﺨﺘﻠﻔﺔ ﻋﻨﺪ اﺷﺘﻘﺎﻗﮭﺎ ﺑﺎﺳﺘﺨﺪام ﻓﯿﻨﯿﻞ-3-ﻣﯿﺜﯿﻞ-5-ﺑﯿﺮازوﻟﻮن. بينما ﺗﺮاوح ﻣﻌﺪل اﻟﻜﺴﺢ ﻣﻦ ١٠٪ إﻟﻰ ٨٨٪ لـ EPS اﻟﻤُﻨﻘﻰ ﺟﺰﺋﯿًﺎ ﻋﻨﺪ ٢٥٠ ﻣﺠﻢ/ﻟﺘﺮ ﺿﺪ ﻛﻞ ﻣﻦ ﺟﺬر 2،2-ﺛﻨﺎئي ﻓﯿﻨﯿﻞ-1-ﺑﯿﻜﺮﯾﻞ ھﯿﺪرازﯾﻞ (DPPH)، ﺟﺬر 2،2'-أزﯾﻨﻮ- ﺛﻨﺎﺋﻲ (3-إﯾﺜﯿﻞ ﺑﻨﺰﯾﻦ-ﺛﯿﺎزوﻟﯿﻦ-6-ﺣﻤﺾ اﻟﺴﻠﻔﻮﻧﯿﻚ) (ABTS)، ﻓﻮق أﻛﺴﯿﺪ اﻟﺪﯾﺴﻤﻮﺗﺎز، ﺟﺬر أﻧﯿﻮن ﻓﻮق اﻷﻛﺴﯿﺪ، ﺑﯿﺮوﻛﺴﯿﺪ اﻟﮭﯿﺪروﺟﯿﻦ، ﺟﺬر اﻟﮭﯿﺪروﻛﺴﯿﻞ، ﻧﺸﺎط ﺧﻠﺐ اﻟﻤﻌﺎدن، وﺗﺜﺒﯿﻂ ﺑﯿﺮوﻛﺴﯿﺪ اﻟﻠﯿﺒﯿﺪ. ﺗﺮاوﺣﺖْ ﻗﺪرة ﻣﻀﺎدات اﻷﻛﺴﺪة ﻟﻠﺤﺪ ﻣﻦ ﻗﻮة ﻣﻀﺎدات اﻷﻛﺴﺪة (FRAP)، ﺗﻘﻠﯿﻞ اﻟﻄﺎﻗﺔ (RP)، وإﺟﻤﺎﻟﻲ ﻣﻀﺎدات اﻷﻛﺴﺪة ﺑﯿﻦ ٧١٤ إﻟﻰ ٢٨٤٨ ﻣﯿﻜﺮوﻏﺮام/ﻣﻞ، أي ﻣﺎ ﯾﻌﺎدل ﻧﺸﺎط ﺣﻤﺾ اﻷﺳﻜﻮرﺑﯿﻚ. ﺑﯿﻨﻤﺎ ﻗُﺪرت EPS اﻟﻤﻨﻘﻰ ﺟﺰﺋﯿًﺎ ﻋﻠﻰ ﺗﺜﺒﯿﻂ ﻧﻤﻮ وﺗﻜﻮﯾﻦ اﻷﻏﺸﯿﺔ اﻟﺤﯿﻮﯾﺔ ﻟﺒﻜﺘﯿﺮﯾﺎ E. coli, S. S. typhimurium، S. aureus و L. monocytogenes ﺑﻤﺘﻮﺳﻂ ﺗﺮاوح ﺑﯿﻦ ٥٣% إﻟﻰ ٧٤٪. ﻛﺎن ﻣﺘﻮﺳﻂ ﺗﺜﺒﯿﻂ ﻛﻞ ﻣﻦ اﻷﻣﯿﻠﯿﺰ، اﻟﺠﻠﻮﻛﻮزﯾﺪاز، اﻟﻜﻮﻟﯿﺴﺘﺮول، واﻹﻧﺰﯾﻢ اﻟﻤﺤﻮل ﻟﻸﻧﺠﯿﻮﺗﻨﺴﯿﻦ ٢٪ إﻟﻰ ٦٦٪، وﻛﺎن ﺗﺜﺒﯿﻂ اﻟﺨﻼﯾﺎ اﻟﺴﺮطﺎﻧﯿﺔ 2-Caco و MCF-7 ٩% إلى ٨٣٪. دﻋﻤﺖ ﺟﻤﯿﻊ ﻋﺪﯾﺪات اﻟﺴﻜﺎرﯾﺪ اﻟﺨﺎرﺟﯿﺔ EPSs ﻧﻤﻮ ﺑﻜﺘﯿﺮﯾﺎ اﻷﻣﻌﺎء اﻟﻤﻔﯿﺪة ﻣﻦ Proteobacteria، Bacteroidetes، Firmicutes و Acinetobacter ﻓﻲ ﺗَﺨﻤﺮ اﻟﺒﺮاز ﻣﻊ إﺟﻤﺎﻟﻲ إﻧﺘﺎج ﻣﻦ SCFA ﯾﺘﺮاوح ﺑﯿﻦ ٤٥٣٠ إﻟﻰ ٦١٠٤ﺟﺰء ﻓﻲ اﻟﻤﻠﯿﻮن. أظﮭﺮت ھﺬه اﻟﺪراﺳﺔ أن EPS اﻟﻤﺴﺘﺨﻠﺺ ﻣﻦ ﻋﺰﻻت ﻣُﺨﺘﺎرة ﻣﻦ LAB ﯾﻤﺘﻠﻚ أﻧﺸﻄﺔ ﺣﯿﻮﯾﺔ ﺟﯿﺪة، وذو طﺒﯿﻌﺔ ﺑﺮﯾﺒﯿﻮﺗﯿﻚ، ﻛﻤﺎ ﻟﮫ ﺗﺄﺛﯿﺮ ﻋﻠﻰ اﻟﻜﺎﺋﻨﺎت اﻟﺤﯿﺔ اﻟﺪﻗﯿﻘﺔ ﻓﻲ اﻷﻣﻌﺎء. ﻋﻼوة ﻋﻠﻰ ذﻟﻚ، ﺗﻌﻤﻞ اﻟﺒﻜﺘﯿﺮﯾﺎ اﻟﻤﻨﺘﺠﺔ ﻟﻠـ EPSﻋﻠﻰ ﺗﺤﺴﯿﻦ وﻗﺖ ﺗَﺠَﻠﺘﻦ ﻣُﺨﻤﱠﺮ اﻟﺤﻠﯿﺐ اﻟﺒﻘﺮي ﻣﻨﺰوع اﻟﺪﺳﻢ. ھﻨﺎك ﺣﺎﺟﺔ إﻟﻰ ﻣﺰﯾﺪ ﻣﻦ اﻟﺪراﺳﺎت ﻟﺪراﺳﺔ ﺗﺄﺛﯿﺮ EPS ﻓﻲ ظﺮوف اﻟﺠﺴﻢ اﻟﺤﻲ واﺳﺘﺨﺪام اﻟﻨﻔﺎﯾﺎت اﻟﺼﻨﺎﻋﯿﺔ ﻹﻧﺘﺎج EPS ﺑﻮاﺳﻄﺔ LAB.
Recommended Citation
Tarique, Mohammed, "EXOPOLYSACCHARIDES OF LACTIC ACID BACTERIA ISOLATED FROM LABNEH: CHARACTERIZATION, BIOACTIVITIES, AND EFFECTS ON GUT MICROBIOME AND FERMENTED MILK RHEOLOGY" (2024). Dissertations. 268.
https://scholarworks.uaeu.ac.ae/all_dissertations/268