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Aerobic growth of Lactobacillus plantarum for production of GOS prebiotics

Young Scientist, Undergraduate winning article by Alice Nield, Food Science graduate, University of Reading

Your gut bacteria can weigh up to 2kg, that is more than the weight of the average human brain. Modulation of the human gut microbiome is associated with many health benefits such as symptom alleviation of Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD) and reductions in liver LDL cholesterol levels.  

Probiotics, live microorganisms (or ‘good bacteria’) that confer health benefits on the host and promote wellbeing,  are found in products such as Yakult and kefir. In this research project, a specific strain of Lactobacillus plantarum  (Optibiotix product LPLDL, see Figure 1) was used. Previously, this had demonstrated the ability to reduce cholesterol in a small human study.  Integral to the activity of the probiotics are the prebiotics: substrates that provide nutrients for beneficial microorganisms and stimulate their growth.  They can be found naturally in products such as Jerusalem artichokes. During this research, galacto-oligosaccharide (GOS) prebiotics were synthesised from the ß-galactosidase enzyme derived from the LPLDL strain of Lactobacillus plantarum.  

Figure 1 Light microscope image of Lactobacillus plantarum (LPLDL) probiotic

under x 40 magnification

It is frequently stated in the media and in literature that there are numerous health benefits from ingesting pre and probiotics. The administration of prebiotics in combination with probiotics is speculated to provide additional benefits, with a synergy occurring within the gut in the form of a synbiotic. In this case, the prebiotic product (the GOS) is targeted to selectively stimulate the growth of the cholesterol reducing probiotic bacteria and improve its survival in the host Gastro-Intestinal (GI) tract, allowing the potential for diet mediated cholesterol reduction in those people with slightly elevated cholesterol levels.

There is a huge challenge in the food industry of producing synbiotics on a large scale due to the use of anaerobic technology for growth of the probiotic species, which is not always available in manufacturing environments. Therefore, challenges in scale up are currently being observed. Part of this research project demonstrated viability of GOS production using aerobically grown probiotics, eliminating the need for anaerobic technology.

LPLDL was grown in MRS broth with lactose for 10 hours both aerobically and anaerobically. After centrifugation and dilution of biomass with sodium phosphate buffer (pH 6.8), cells were lysed mechanically by bead beating to yield intracellular ß-galactosidase crude enzyme, see Figure 2.

Figure 2 Methodology for production of ß-galactosidase enzyme for production of

GOS

This enzyme was used to catalyse the production of GOS via the transgalactosylation reaction using lactose as the substrate.  GOS are prebiotic carbohydrates comprised of 2-8 saccharide units, with one of these being a terminal glucose or galactose unit. GOS selectively stimulate Bifidobacterium and Lactobacillus at the expense of harmful bacteria such as Staphylococcus aureus, see Figure 3.

Figure 3 Illustrating the kinetically controlled synthesis reaction for GOS from lactose, with the hydrolysis reaction occurring simultaneously to yield D-galactose and
D-glucose

GOS were synthesised over 36 hours, using 20 different temperature, pH and lactose concentration conditions. As lactose was consumed, it was either converted to GOS or hydrolysed to glucose and galactose (its constituent monosaccharides). GOS yields peaked between 13 and 17 hours, before glucose and galactose increased. ​Anaerobically, GOS yields of 36% were achieved. There was a clear compromise necessary between a high enough temperature to allow for lactose solubility yet ensuring enzyme activity was maintained throughout. 

When grown aerobically, ß-galactosidase was produced by the LPLDL strain and GOS were synthesised albeit with lower maximum yields of 15%. This is promising for the prebiotics industry, suggesting that use of facultative anaerobic probiotics that respond well to oxidative stress could be used to produce a prebiotic, when using probiotics grown in either anaerobic or aerobic conditions. Conditions could be altered to achieve higher yields or comparable yields to that of the anaerobic growth, such as varying growth time, temperature or lactose concentration to match the growth characteristics of the probiotic under aerobic conditions.

In conclusion, this research project demonstrated that GOS were produced from enzymes derived from aerobic growth of L. plantarum, posing significant improvements to current challenges in the food industry relating to anaerobic growth. This would allow for the scale up of this synbiotic technology to yield a viable product with the potential for selectively increasing probiotics with known cholesterol reducing abilities, so that cholesterol could be reduced using diet mediated strategies in those people with slightly elevated cholesterol levels.

We probably all know someone with elevated cholesterol levels, or perhaps some of you reading this have been told by your doctor to reduce your cholesterol levels. Cardiovascular disease (CVD) is the 2nd largest cause of mortality in the UK and with high LDL cholesterol being a main risk factor, the ability to scale up this technology in industry would allow for potential reductions in cholesterol before medication becomes a necessity. Whilst reading this article, at least one person in the UK will have passed away from CVD. This puts into perspective the impact this disease has both in the UK and globally. The scale up of this technology using readily available aerobic technology would be a stepping stone in both improving gut microbiology composition and reducing elevated cholesterol levels within the population (see Figure 4).

Figure 4 Strategy for improving gut microbiology composition and reducing elevated
cholesterol levels

With thanks to Professor Bob Rastall and Dr Vasiliki Kachrimanidou (University of Reading) for their supervision and support and to Optibiotix for their sponsorship of this research project.

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