Originally publish in Food NZ magazine by Director of the NZ Food Safety Science and Research Centre Dr Catherine McLeod, ESR Senior Scientist Dr Lucia Rivas, ESR Science Leader Dr Angela Cornelius, and science writer Glenda Lewis.
People used to think of all bacteria as inherently bad for humans, a notion successfully fostered by marketers of cleaning products. At the NZ Food Safety Science & Research Centre (the Centre) the baddies get most of the attention, but scientists increasingly find that the community of microorganisms inside our gut (our microbiome) are essential for good health, including our mental health. We need them as much as they need us. The implications for the food industry are enormous.
Diet is key to a healthy gut microbiome Principal scientist at Fonterra, Dr Shalome Bassett, said in a recent profile published by the Centre, “From birth, the microbes in your gut are helping to train your immune system, effectively programming it for life. They have an extraordinary influence on our long-term health and development, including digestion, metabolism, and the production of neurotransmitters that affect behaviour and cognitive function. There’s new evidence to suggest that this process can start in the womb. Our gut microbiome undergoes dynamic changes in its make-up and
diversity over our lifespan, particularly in childhood. Some data suggest that alterations in the gut microbiome in young children can lead to an increased risk of developing diseases later in life, such as asthma and Crohn’s disease. Diet is absolutely key to a healthy gut microbiome”.
No wonder then that educated, health-conscious consumers have quickly latched on to these emerging findings, and are enthusiastically embracing foods and drinks fermented by ‘good’ bacteria and containing live bacteria when consumed. Examples are kombucha, kimchi, kefir, yoghurt and cheese. Hundreds, if not thousands, of amateur sourdough bakers emerged from lockdown New Zealand though many concluded that this is an art best left to the professionals.
Those foods and drinks containing specific strains of microorganisms proven to be of benefit to our health – and present in sufficient amounts to provide the proven health benefit – are called probiotics.
The expectation is that ingesting certain bacteria will, at least in the short term, help to improve the balance, diversity and overall health of the gut microbiota.
Booming demand for probiotics
The naturally-fermented food category, including probiotics, is booming and expected to expand by a further $60bn to $700bn in the next two or three years. New Zealand is after its share and well placed to win it.
Thanks to a new way of using whole genome sequencing to verify the identity and safety of lactic acid bacteria (LAB), developed under the auspices of the Centre by Fonterra, ESR and Massey University scientists, the commercialisation of new LAB strains for use as probiotics (or in fermented foods) has become much faster and cheaper.
LAB strains are used widely by the food industry as starter cultures but also as adjunct cultures, contributing to the flavour, texture, and nutritional value of fermented foods. Lactic acid is a by-product of the bacteria’s digestion – it gives yoghurt its tangy taste and stops the growth of other microorganisms, preventing spoilage and thus lengthening shelf life. Not surprisingly, LABs are the dominant probiotic species globally. The hundreds of wonderful cheese varieties owe their characteristic textures and flavours to the different LABs and other microorganisms used in their making. LABs are the invisible, unpaid, yet crucial workforce of the dairy industry. And they also play an important role in our development from birth.
Shalome says, “LABs, in particular the Bifidobacteria strains, are naturally present in human breast milk and are the early colonisers of the infant gut, helping it to mature and develop normally. The infant gut is leaky at birth and the right bacteria help the gaps in the gut to ‘close’. This is important for priming our immune system for life-long health and wellness.”
Fonterra continues to search for new LAB strains (they occur naturally in the environment, particularly milk) to add to its enormous collection of over forty thousand. They keep their library of isolates at temperatures below minus 80ºC, preserved in glycerol or milk. Many have yet to be investigated for possible commercialisation. As well as using LABs for their own range of dairy products, they manufacture and sell probiotic LAB strains to other producers – a lucrative sideline.
Fonterra discovered and owns two of the top five probiotic strains in the world – Lacticaseibacillus rhamnosus HN001™ (also known as DR20® and Surestart LactoB™) and Bifidobacterium animalis HN019™ (also known as DR10® and Surestart BifidoB™)]. More than 100 scientific Booming demand for probiotics The naturally-fermented food category, including probiotics, is booming and expected to expand by a further $60bn to $700bn in the next two or three years. New Zealand is after its share and well placed to win it.
Thanks to a new way of using whole genome sequencing to verify the identity and safety of lactic acid bacteria (LAB), developed under the auspices of the Centre by Fonterra, ESR and Massey University scientists, the commercialisation of new LAB strains for use as probiotics (or in fermented foods) has become much faster and cheaper. LAB strains are used widely by the food industry as starter cultures but also as adjunct cultures, contributing to the flavour, texture, and nutritional value of fermented foods.
Lactic acid is a by-product of the bacteria’s digestion – it gives yoghurt its tangy taste and stops the growth of other microorganisms, preventing spoilage and thus lengthening shelf life. Not surprisingly, LABs are the dominant probiotic species globally. The hundreds of wonderful cheese varieties owe their characteristic textures and flavours to the different LABs and other microorganisms used in their making. LABs are the invisible, unpaid, yet crucial workforce of the dairy industry. And they also play an important role in our development from birth.
Shalome says, “LABs, in particular the Bifidobacteria strains, are naturally present in human breast milk and are the early colonisers of the infant gut, helping it to mature and develop normally. The infant gut is leaky at birth and the right bacteria help the gaps in the gut to ‘close’. This is important for priming our immune system for life-long health and wellness.”
Fonterra continues to search for new LAB strains (they occur naturally in the environment, particularly milk) to add to its enormous collection of over forty thousand. They keep their library of isolates at temperatures below minus 80ºC, preserved in glycerol or milk. Many have yet to be investigated for possible commercialisation. As well as using LABs for their own range of dairy products, they manufacture and sell probiotic LAB strains to other producers – a lucrative sideline.
Fonterra discovered and owns two of the top five probiotic strains in the world – Lacticaseibacillus rhamnosus HN001™ (also known as DR20® and Surestart LactoB™) and Bifidobacterium animalis HN019™ (also known as DR10® and Surestart BifidoB™). More than 100 scientific studies have been published on these strains with clinical evidence to support a wide range of human health benefits, including improved immunity, gut comfort, reduced incidence of postnatal depression and gestational diabetes, and reduced eczema in infants. These strains are used globally in a wide range of probiotic supplements and food products such as infant formula.
To Fonterra’s knowledge, it is the only dairy company in the world which manufactures its own starter cultures. It has a complete system, and therefore control, from collection and testing of strains to final product.
Strict regulatory control
Precise identification of strains, and a guarantee of their safety for human consumption, is paramount when it comes to producing bacteria to make or add to food. The global regulations around probiotics are extremely strict. Shalome says, “Probiotic preparations must contain live bacteria in defined doses, with specifically identified human health benefits. The World Health Organisation definition is: ‘live microorganisms, which when administered in adequate amounts, confer a health benefit on the host.’ The number of bacteria required for a positive health effect must be proven in human clinical trials.”
Whole genome sequencing (WGS) technology can differentiate strains down to the last mutation. But the name is somewhat misleading in practice, as the size of bacteria genomes and the repetitions of certain sequences within the genome, mean that only segments of the DNA are in fact sequenced or ‘read’, resulting in a genome that is fragmented.
Scientists can home in on different bits (key genes or tell-tale mutation sites) that serve to identify a strain. The construction of genomes with only small amounts of fragmentation is relatively expensive but is required to verify safety for human consumption and ensure there are no known traits for antibiotic resistance, virulence or toxicity. In the case of LABs, it can also be used to identify potential health benefit traits or to unlock new tastes or textures in fermented foods. Although the current regulatory requirement is for fewer than 500 segments, Fonterra want fewer than 50 segments to ensure their LAB genomes are the best quality possible.
Advances in gene sequencing technologies
Routine sequencing at ESR and other research laboratories in New Zealand is most commonly carried out on an Illumina machine, and is what they call ‘short-read’ (short sequences of DNA in 150-250 base pair pieces). It’s fast and accurate but generates very fragmented genomes.
It is good enough for the purposes of identification, surveillance and tracking of potential pathogens, which is usually all that is required by industry. By comparison, 'long-read’ sequencing yields much longer continuous sequences of 10,000 to 100,000 base pairs and so is much more complete.
The different sequencing machines on the market all differ in price, speed, number of samples that can be processed at a time, and the number of continuous base pairs they read (short- vs long-read).
Horses for courses
Until recently, Fonterra had to send DNA from bacterial isolates intended for use as probiotics overseas, for long-read sequencing on a PacBio machine, to give them the resolution they needed to meet safety regulations. These are expensive machines, require large quantities of high-quality DNA, and are not available in New Zealand. PacBio sequencing was taking about eight weeks, and often the sample degraded in transit, meaning multiple preparations of DNA were being prepared and sent overseas. Months would go by.
Frustrated by these problems, Fonterra approached ESR and the Centre to see if they could come up with a local solution. Shalome was aware that ESR now had MinION long-read sequencers and was evaluating their use for whole genome sequencing of bacteria. It had already become invaluable for sequencing and tracing positive Covid-19 samples.
The MinION is a small handheld device developed by Oxford Nanopore Technologies Ltd, which requires less DNA and is much cheaper than the ‘Rolls Royce’ PacBio. Early versions were less accurate, however recent technological advancements have significantly improved data quality to about the same level.
The idea ESR came up with was to crossmap MinION readouts with Illumina short-read data to create a near-complete genome. Generating a hybrid genome sequence using both short- and long-read data sets compensates for any reading errors. The long pieces of DNA sequence generated by MinION create a scaffold that the short, but more accurate, Illumina-generated sequences can be aligned to, thus helping to bridge any gaps.
Crucially, scientists at Massey University knew how to get high quality DNA from LABs following another project they’d done with Fonterra, so all three organisations decided to work in partnership on this initiative.
Fonterra wanted to test the accuracy of Illumina-MinION hybrids against five already sequenced LAB strains (Illumina-PacBio hybrids) and contributed the 60% industry funding required. The ultimate goal was to develop long-read sequencing capability in New Zealand for use by all New Zealand food companies. On this basis, Fonterra and the Centre pulled together a research funding application and secured the balance of 40 percent funding from the Ministry for Business, Innovation and Employment.
Success!
The results exceeded all hopes. The research team established an efficient workflow where DNA is extracted from the LAB of interest, sequenced on the MinION, and the data combined with short-read sequencing data previously obtained using Illumina technology. The team was able to build hybrid genomes that were equivalent to (and in some cases better than) the Illumina-PacBio hybrids.
These high quality, near-complete genomes were suitable for safety assessment of LABs at a quarter of the cost of using PacBio and with a much quicker turn-around-time. The data and modus operandi were checked and confirmed by independent international collaborators who are world experts in LAB genomics. This was key to quality assurance.
The workflow has been successfully implemented at Fonterra, ESR and Massey University to ensure the method is widely available for use by the New Zealand food industry for similar applications. This small study was so successful that Fonterra extended the number of strains sequenced on the MinION.
Shalome says, “After the initial 5 isolates were tested, over 300 more were sequenced, with 90% of our Illumina-MinION hybrid genome assemblies having fewer than 10 gaps in their sequence. Overall 20 percent of these strains now have complete genomes with no gaps at all. We were absolutely delighted with the results.”
ESR is now using MinION’s higher capacity big brother GridION, so that they can run more samples at a time. Shalome says ESR – namely Dr Angela Cornelius and Dr Lucia Rivas – have been excellent to work with, and everyone is delighted with the outcome. Lynn Rogers from Massey University has also been an important member of the project team.
Combining resources has enormous benefits
Dr Catherine McLeod, director of the Centre, says this project is a prime example of what can be achieved when industry, science and government put their heads and limited resources together.
“Catalysing this kind of joint research collaboration is what the Centre is here to do. At the heart of it all is greater food safety assurance and protection of New Zealand’s reputation, as well as better health.”
Shalome is excited about the potential of probiotics as a natural way to promote and support mental and physical health and wellbeing.
“Given that 70 percent of the body’s immune reactions occur in the gut, and the knowledge that changes to the bacteria present in the gut are associated with health issues such as irritable bowel syndrome (IBS), anxiety and depression – all now so sadly prevalent both globally and in New Zealand – developing safe, targeted food solutions (including probiotics) is critical. We believe that dairy-based solutions, such as our proprietary probiotic strains, can really make a difference to improving people’s health. Knowing we can be part of the solution certainly gets me up in the morning.”
This article was transcribed with permission from Peppermint Press.
McLeod, C., Rivas, L., Cornelius, A., & Lewis, G. (2022). Creating a 'hybrid' whole genome sequence – A clever local solution to testing the safety of bacteria used in the food industry. FoodNZ, 22(1), 18–21.
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