Isolation and Selection of Cholesterol-lowering and y-Aminobutyric Acid-producing Lactic Acid Bacteria for Producing Functional Fermented Dairy Products

Abstract

Nowadays, functional foods are popular as high demand of consumers. The use of starters) from lactic acid bacteria (LAB) for functional food productions is one of alternative ways to produce functional foods. This research collected 60 samples of various fermented foods and raw milk in Thailand to isolate LAB. A total of 244 strains from 39 fermented foods were primarily screened using basal medium containing 100 ug/ml cholesterol and 0.1% yeast extract and found 139 strains survival. However, for secondary screening, only 10 strains survived in the same medium that yeast extract was removed. Five LAB strains (ENM104, ENM203, ENM204, NNM205 and NNM304) were passed tertiary screening as their proliferations. Of these, only ENM104 reduced cholesterol at 7.53 $ 1.78% in basal medium supplemented with 100 ug/ml cholesterol-PEG600 at 72 h of fermentation. Screening of GABA producer, ten LAB isolates obtained from secondary screening found that only SPS109 strain in MRS fortified with 5 mg/ml monosodium glutamate (MSG) released 1{157.01 4.76 g/ml at 72 h of incubation. Selected LAB strains were identified using both biochemical test with 49 sugar fermentations of API 50 CHL test kit and molecular identification with 16S TRNA gene. Strains ENM104 and SPS109 showed the highest similarity with Pediococcus pentosaceus DSM20336' at 99.65 % and Lactobacillus plantarum DSM201747 at 99.93%, respectively. Both LAB strains were investigated for probiotic properties. The results showed that none of strains hydrolysed starch, but only SPS109 hydrolysed tributyrin. However, both LAB strains hydrolysed casein. Based on bile salt hydrolysis, both strain hydrolysed glycocholic acid (GCA), taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Gastrointestinal tolerance was tested in vitro displayed that both strains survived in acidic condition mimicking from stomach with viable at 6.20 ÷ 0.12 and 7.39 ÷ 0.12 log CFU/ml (initial cells at 10.18 * 0.02 and 10.46 $ 0.04 log CFU/ml) of ENMI04 and SPS109, respectively. Furthermore, cells slightly increased in basic condition mimicking from intestine at 6.49 # 0.00 and 7.63 = 0.02 log CFU/ml, respectively. Cholesterol-lowering property testing in MRS containing 100 ug/ml of cholesterol-PEG600 found that ENM104 and SPS109 reduced 15.34 # 1.12 and 10.05 ÷ 2.32% at 72 h of incubation, respectively. After that, ENM104 in the same medium produced cholesterol oxidase and its specific activity was 8.73 = 2.00 mU/mg protein after 72 h of incubation. ENM104 and SPS109 strains tolerated in 1 mM of H202 for 20 and 12 h, respectively. Antioxidant activity was also tested in MRS by ORAC in umol TE/ml found 37.26 ÷ 0.77 at 48 h for ENM104 and 34.01 # 3.30 at 72 h for SPS109. A similar result was observed for total phenolic content (TPC) at 72 h of incubation by both strains as 0.67 $ 0.00 and 0.68 = 0.00 umol GE/ml. For application in dairy products, single and co-culture (1:1) in milk/whey fermentation was investigated. In fermented milk, it found the lowest pH at 5.32 ÷ 0.01 in co-culture set at 72 h, and this set had population in log CFU/ml of ENM104 at 6.93 = 0.01 and SPS109 at 8.27 # 0.04. After 72 h incubation, cholesterol was reduced in fermented milk at 15.71 # 0.64% for ENM104 set and 10.98 ÷ 3.80% for co-culture set. On the other hand, GABA was found at 4.57 # 0.01 and 4.47 # 0.04 ug/ml in SPS109 and co-culture sets, respectively. A co-culture set showed maximum at 57.63 # 2.97 % inhibition against on angiotensin-converting enzyme (ACE) as cause of hypertensive blood pressure by this enzyme. The highest antioxidant activity based on ORAC and TPC was found at 24 h incubation in the co-culture set at 2.58 = 0.26 umol TE/ml at 24 h and TPC at 0.31 ÷ 0.03 umol GE/ml. In fermented whey, the lowest pH at 4.35 ÷ 0.01| was observed in a co-culture set with LAB population (log CFU/ml) at 7.19 ÷ 0.03 for ENM104 and 8.41 # 0.02 for SPS 109. ENM104 set maximally reduced 54.86 ÷ 13.48 % cholesterol at 72 h. However, GABA content reported in ug/ml in SPS109 set (4.84 # 0.01) was higher than a co-culture set (4.25 # 0.03). The highest ACE inhibition at | 33.95 $ 4.95 % was found in a co-culture set at 72-h incubation. This set also showed the maximum antioxidant activity both in ORAC and TPC at 0.86 * 0.22 umol TE/ml and 0.06 = 0.01 umol GE/ml. Overall result prove that both LAB strains have potential to be used as starters for producing functional fermented dairy products.