Abstract
In this study, thermo-tolerant strain of Lactobacillus bulgaricus (L. bulgaricus) was developed using gradual increase in temperature to induce Adaptive Laboratory Evolution (ALE). Viable colony count of 1.87 ± 0.98 log cfu/mL was achieved at 52 °C, using MRS agar supplemented with 2% lactose. Changes in bacteria morphology were discovered, from rod (control) to filament (52 °C) to cocci after frozen storage (−80 °C). When milk was inoculated with thermo-tolerant L. bulgaricus, lactic acid production was absent, leaving pH at 6.84 ± 0.13. This has caused weakening of the protein network, resulting in high whey separation and lower water-holding capacity (37.1 ± 0.35%) compared to the control (98.10 ± 0.60%). Significantly higher proteolytic activity was observed through free amino acids analysis by LC-MS. Arginine and methionine (237.24 ± 5.94 and 98.83 ± 1.78 µg/100 g, respectively) were found to be 115- and 275-fold higher than the control, contributing to changing the aroma similar to cheese. Further volatile analysis through SPME-GC-MS has confirmed significant increase in cheese-aroma volatiles compared to the control, with increase in diacetyl formation. Further work on DNA profiling, metabolomics and peptidomics will help to answer mechanisms behind the observed changes made in the study.
Highlights
In yoghurt making, lactic acid bacteria (LAB) use the lactose in milk and produce lactic acid through fermentation
Starting from the isolated pure culture of L. bulgaricus grown at 42 ◦ C, temperature elevation was used as an environmental stimulus to induce Adaptive Laboratory Evolution (ALE)
Over the repeated exposure to elevated growth temperature over time, the thermo-tolerant strain survived to 52 ◦ C albeit a clear change in morphology and reduced production of lactic acid during fermentation
Summary
Lactic acid bacteria (LAB) use the lactose in milk and produce lactic acid through fermentation. The pH is reduced from 6.8 to 4.5, resulting in the formation of milk gel network with changes in the casein structure. Factors such as type of LAB strain, incubation time and temperature during fermentation play a considerable role in stabilising the interactions of casein micelles and forming a three-dimensional gel network [1]. Some LAB strains used in yoghurt-making are considered probiotics, as they may help the gut microbiome and overall health. This has caused an increase in consumption and demand for probiotic dairy products in recent years [4]. In the U.S, the FDA requires dairy product fermentation to be done with two particular bacteria: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) [4]
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