Nutritional and Volatile Characterisation of Milk Inoculated with Thermo-Tolerant Lactobacillus bulgaricus through Adaptive Laboratory Evolution

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.

Adaptive Laboratory Evolution to obtain lactic acid bacteria strains of industrial interest - a review

“…ALE was used as a tool to mainly improve or adapt LAB toward an increase in lactic acid production capacity, an increase in survival rate at acidic pH values, a better tolerance to different temperature conditions, a higher vitamin production, the maintenance of genetic stability of the strains under multiple stresses, and the investigation of how antibiotics can result in adaptations within the LAB genome. Among the different ways that ALE can be applied (Figure 2), it was observed that, of the 12 studies cited, seven utilized batch mode ALE (Zhang et al, 2012;Chen et al, 2015;Ming et al, 2016;Kwon et al, 2018;Prasad et al, 2020;Liang et al, 2021;Liu et al, 2021), one utilized the fed-batch mode method (Singhvi et al, 2018), and one utilized batch, fed-batch, and pulse fed-batch modes (Mladenović et al, 2019). The remaining works used distinct methods that are not described in Figure 2.…”

“…Cellular Division of these microorganisms can occur rapidly (20 min to 10 h/generation), for a few generations (100 to 500) (Sandberg et al, 2019). Thus, batch mode experiments are simple and the most widely used protocols for ALE and have been used in most of the articles cited in this review (Table 1) (Zhang et al, 2012;Chen et al, 2015;Ming et al, 2016;Kwon et al, 2018;Singhvi et al, 2018;Mladenović et al, 2019;Prasad et al, 2020;Liang et al, 2021;Liu et al, 2021).…”

“…During fermentation, these microorganisms are subjected to stress conditions due to factors such as temperature, pH, and osmotic pressure. For example, an increase in osmotic pressure across the bacterial cell wall occurs at the start of the fermentation process due to the high concentration of solutes in the medium, resulting in water efflux, dehydration, and consequent inactivation of enzymes relevant to the metabolism of these microorganisms (Mitchell et al, 2009;Liang et al, 2021).…”

“…In short, ALE allows the production of microbial strains for the production of fermented foods with unique sensory characteristics and/or that provide significant health benefits to consumers (Steele et al, 2013;Rakhmanova et al, 2018). Several studies have been performed using this method to improve industrial LAB strains, such as increased lactic acid production (Zhang et al, 2012;Chen et al, 2015;Ju et al, 2016;Mladenović et al, 2019;Liang et al, 2020), thermotolerance (Chen et al, 2015;Kwon et al, 2018;Liang et al, 2021), multiple-stress tolerance (Ming et al, 2016), increased vitamin production (Liu et al, 2021) and the production of aromatic compounds of industrial interest (Liang et al, 2021).…”

The Antioxidants Improvement of Yogurt with Three Sources of Non-Alcohol Anthocyanin Extract and Metroxylon sagu as a Natural-Based Thickener

“…Furthermore, the bonds between protein and fat affected viscosity; changes in casein milk with the same hydrophilic properties as other protein types caused increased viscosity [55]. Fermentation of lactose produces lactic acid, which plays a role in milk protein to produce a unique gel-like texture and odor in yogurt [56].…”