Hielscher Ultrasound Technology

Biosynthetic Production of Human Milk Oligosaccharides

The biosynthesis of human milk oligosaccharides (HMOs) via fermentation or enzymatic reactions is a complex, consuming and often low-yielding process. Ultrasonication increases the mass transfer between substrate and cell factories ans stimulates cell growth and metabolism. Thereby, sonication intensifies fermentation and bio-chemical processes resulting in an accelerated and more efficient production of HMOs.

Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs), also known as human milk glycans, are sugar molecules, that are part of the oligosaccharides group. Prominent examples of HMOs include 2’-fucosyllactose (2′-FL), lacto-N-neotetraose (LNnT), 3’-galactosyllactose (3′-GL), and difucosyllactose (DFL).
While human breast milk iscomposed of more than different 150 HMO structures, only 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) are currently produced on commercial level and used as nutritional additives in infant formula.
Human milk oligosaccharides (HMOs) are known for their significance in baby nutrition. Human milk oligosaccharides are a unique type of nutrients, which act as prebiotics, anti-adhesive antimicrobials, and immunomodulators within the infant’s gut and contribute substantially to the brain development. HMOs are exclusively found in human breast milk; other mammalian milks ( e.g. cow, goat, sheep, camel etc.) do not have these specific form of oligosaccharides.
Human milk oligosaccharides are the third most abundant solid component in human milk, which can be present either in dissolved or emulsified or suspended form in water. Lactose and fatty acids are the most abundant solids found in human milk. HMOs are present in a concentration of 0.35–0.88 ounces (9.9–24.9 g)/ L. Approximately 200 structurally different human milk oligosaccharides are known. The dominant oligosaccharide in 80% of all women is 2′-fucosyllactose, which is present in human breast milk at a concentration of approximately 2.5 g/ L.
Since HMOs are not digested, they do not calorically contribute to nutrition. Being indigestible carbohydrates, they function as prebiotics and are selectively fermented by desirable gut microflora, especially bifidobacteria.

Health Benefits of Human Milk Oligosaccharides (HMOs)

  • promote the development of infants
  • are important for brain development
  • has anti-inflammatory and
  • anti-adhesive effects in the gastro-intestinal tract
  • supports the immune system in adults
Ultrasonication and the use of ultrasonic bioreactors (sono-bioreactors) are highly effective to promote mass transfer between substrate and living cells used as cell factories

The ultrasonic processor UIP2000hdT increases mass transfer and activates cell factories for higher yields of biosynthesized biological molecules such as HMOs

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Biosynthesis of Human Milk Oligosaccharides

Cell factories and enzymatic / chemo-enzymatic systems are current technologies used for the synthesis of HMOs. For HMO production on industrial scale, the fermentation of microbial cell factories, bio-chemical synthesis, and different enzymatic reactions are feasible ways of HMO bio-production. Due to economic reasons, the bio-synthesis via microbial cell factories is currently the only technique used on industrial production level of HMOs.

Fermentation of HMOs using Microbial Cell Factories

E.coli, Saccharomyces cerevisiae and Lactococcus lactis are commonly used cell factories used for the bio-production of biological molecules such as HMOs. Fermentation is a biochemical process using micro-organisms to convert a substrate into targeted biological molecules. Microbial cell factories use simple sugars as substrate, which they convert into HMOs. Since simple sugars (e.g. lactose) are an abundant, cheap substrate, this keeps the bio-synthesis process cost-efficient.
Growth and bioconversion rate are mainly influenced by the mass transfer of nutrients (substrate) to the microorganisms. The mass transfer rate is a main factor that affects the product synthesis during fermentation. Ultrasonication is well known to promote mass transfer.
During fermentation, the conditions in the bioreactor must be constantly monitored and regulated so that the cells can grow as quickly as possible in order to then produce the targeted biomolecules (e.g. oligosaccharides such as HMOs; insulin; recombinant proteins). Theoretically, the product formation starts as soon as the cell culture begins to grow. However especially in genetically modified cells such as engineered microorganisms it is usually induced later by adding a chemical substance to the substrate, which upregulates the expression of the targeted biomolecule. Ultrasonic bioreactors (sono-bioreactor) can be precisely controlled and allow for the specific stimulation of microbes. This results in an accelerated biosynthesis and higher yields.
Ultrasonic lysis and extraction: Fermentation of complex HMOs might be limited by low fermentation titers and products remaining intracellular. Ultrasonic lysis and extraction is used to release intracellular material before purification and down-stream processes.

Ultrasonically Promoted Fermentation

The growth rate of microbes such as Escherichia coli, engineered E.coli, Saccharomyces cerevisiae and Lactococcus lactis can be accelerated by increasing the mass transfer rate and cell wall permeability by applying controlled low-frequency ultrasonication. As a mild, non-thermal processing technique, ultrasonication applies purely mechanical forces into the fermentation broth.
Acoustic Cavitation: The working principle of sonication is based on acoustic cavitation. The ultrasonic probe (sonotrode) couples low-frequency ultrasound d waves into the medium. The ultrasound waves travel through the liquid creating alternating high-pressure (compression) / low-pressure (rarefaction) cycles. By compressing and stretching the liquid in alternating cycles, minute vacuum bubbles arise. These small vacuum bubbles grow over several cycles until they reach a size where they cannot absorb any further energy. At this point of maximum growth, the vacuum bubble implodes violently and generates locally extreme conditions, known as the phenomenon of cavitation. In the cavitational “hot-spot”, high pressure and temperature differentials and intense shear forces with liquid jets of up to 280m/sec can be observed. By these cavitational effects, thorough mass transfer and sonoporation (the perforation of cell walls and cell membranes) is achieved. The nutrients of the substrate are floated to and into the living whole cells, so that the cell factories are optimally nourished and growth as well as conversion rates are accelerated. Ultrasonic bioreactors are a simple, yet highly effective strategy to process biomass in a one-pot biosynthesis process.
A precisely controlled, mild sonication is well-known to intensify fermentation processes.
Sonication improves “the productivity of many bioprocesses involving live cells via the enhancement of substrate uptake, enhanced production or growth by increasing cell porosity, and potentially enhanced release of cell components.” (Naveena et al. 2015)
Read more about ultrasonically-assisted fermentation!
Advantages of Ultrasonically Intensified Fermentation

  • Increased Yield
  • Accelerated Fermentation
  • Cell-Specific Stimulation
  • Enhanced Substrate Uptake
  • Increased Cell Porosity
  • Easy-to-Operate
  • Safe
  • Simple Retro-Fitting
  • Linear Scale-up
  • Batch or InIine Processing
  • Fast RoI

Naveena et al. (2015) found that ultrasonic intensification offers several advantages during bioprocessing, including low operating costs compared to other enhancing treatment options, simplicity of operation and modest power requirements.

Agitated ultrasonic tank (sono-bioreactor) for batch processing

Tank with 8kW ultrasonicators and agitator

High-Performance Ultrasonic Fermentation Reactors

Fermentation processes involve living microorganisms such as bacteria or yeast, which function as cell factories. Whilst sonication is applied to promote mass transfer and increase microorganism’s growth and conversion rate, it is crucial to control the ultrasonic intensity precisely in order to avoid the destruction of the cell factories.
Hielscher Ultrasonics is specialist in designing, manufacturing and distributing high-performance ultrasonicators, which can be precisely controlled and monitored to ensure superior fermentation yields.
Precise control over the ultrasonic process parameters by Hielscher Ultrasonics' intelligent softwareProcess control is not only essential for high yields and superior quality, but enables to repeat and reproduce outcomes. Especially when ist comes to the stimulation of cell factories, the cell-specific adaptation of the sonication parameters is essential to achieve high yields and to prevent cell degradation. Therefore, all digital models of Hielscher ultrasonicators are equipped with intelligent software, which allows you to adjust, monitor, and revise sonication parameters. Ultrasonic process parameters such as amplitude, temperature, pressure, sonication duration, duty cycles, and energy input are essential to promote HMO production via fermentation.
The smart software of Hielscher ultrasonicators records automatically all important process parameters on the integrated SD-card. The automatic data recording of the sonication process are the foundation for process standardization and reproducibility / repeatability, which are required for Good Manufacturing Practices (GMP).

Hielscher Ultrasonics Cascatrode

CascatrodeTM in an ultrasonic flow cell reactor

Ultrasonic Rectors for Fermentation

Hielscher Ultrasonics CascatrodeHielscher offers ultrasonic probes of various size, length and geometries, which can be used for batch as well as continuous flow-through treatments. Ultrasonic reactors, also known as sono-bioreactors, are available for any volume covering the ultrasonic bioprocessing from small lab samples to pilot and fully-commercial production level.
It is well known that the location of the ultrasonic sonotrode in the reaction vessel influences the distribution of cavitation and micro-streaming within the medium. Sonotrode and ultrasonic reactor should be chosen in accordance to the processing volume of the cell broth. Whilst sonication can be performed in batch as well as in continuous mode, for high production volumes the use of a continuous flow installation is recommended. Passing through an ultrasonic flow cell, all cell medium gets exactly the same exposure to sonication ensuring the most effective treatment. Hielscher Ultrasonics broad range of ultrasonic probes and flow cell reactors allows to assemble the ideal ultrasonic bioprocessing setup.

Hielscher Ultrasonics – From Lab to Pilot to Production

Hielscher Ultrasonics covers the full spectrum of ultrasonic equipment offering compact hand-held ultrasonic homogenisers for sample preparation to bench-top and pilot systems as well as powerful industrial ultrasonic units that easily process truckloads per hour. Being versatile and flexible in installation and mounting options, Hielscher ultrasonicators can be easily integrated into all kinds of batch reactors, fed-batches or continuous flow-through setups.
Various accessories as well as customized parts allow for the ideal adaptation of your ultrasonic setup to your process requirements.
Built for 24/7 operation under full load and heavy duty in demanding conditions, Hielscher ultrasonic processors are reliable and require only low maintenance.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Batch Volume Flow Rate Recommended Devices
1 to 500mL 10 to 200mL/min UP100H
10 to 2000mL 20 to 400mL/min UP200Ht, UP400St
0.1 to 20L 0.2 to 4L/min UIP2000hdT
10 to 100L 2 to 10L/min UIP4000hdT
n.a. 10 to 100L/min UIP16000
n.a. larger cluster of UIP16000

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Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers for dispersion, emulsification and cell extraction.

High-power ultrasonic homogenizers from lab to pilot and industrial scale.

Literature / References

Facts Worth Knowing

Biosynthesis using Cell Factories

A microbial cell factory is a method of bioengineering, which utilizes microbial cells as a production facility. By genetically engineering microbes, the DNA of microorganisms such as bacteria, yeasts, fungi, mammalian cells, or algae is modified turning microbes into cell factories. Cell factories are used to convert substrates into valuable biological molecules, which are used e.g. in food, pharma, chemistry and fuel production. Different strategies of cell factory-based biosynthesis aim at the production of native metabolites, expression of heterologous biosynthetic pathways, or protein expression.
Cell factories can be used to either synthesize native metabolites, to express heterologous biosynthetic pathways, or to express proteins.

Biosynthesis of native metabolites

Native metabolites are defined as biological molecules, which the cells used as cell factory produce naturally. Cell factories produce these biological molecules either intracellularly or a secreted substance. The latter is preferred since it facilitates the separation and purification of the targeted compounds. Examples for native metabolites are amino and nucleic acids, antibiotics, vitamins, enzymes, bioactive compounds, and proteins produced from anabolic pathways of cell.

Heterologus Biosynthetic Pathways

When trying to produce an interesting compound, one of the most important decisions is the choice of production in the native host, and optimize this host, or transfer of the pathway to another well-known host. If the original host can be adapted to an industrial fermentation process, and there are no health-related risks in doing so (e.g., production of toxic by-products), this can be a preferred strategy (as was the case e.g., for penicillin). However, in many modern cases, the potential of using an industrially preferred cell factory and related platform processes out-weighs the difficulty of transferring the pathway.

Protein Expression

The expression of proteins can be achieved via homologous and heterologous ways. In homologous expression, a gene that is naturally present in an organism is over-expressed. Through this over-expression, a higher yield of a certain biological molecule can be produced. For heterologous expression, a specific gene is transferred into a host cell in that the gene is not present naturally. Using cell engineering and recombinant DNA technology, the gene is inserted into the host’s DNA so that the host cell produces (large) amounts of a protein that it would not produce naturally. Protein expression is done in a variety of hosts from bacteria, e.g. E. coli and Bacillis subtilis, yeasts, e.g., Klyuveromyces lactis, Pichia pastoris, S. cerevisiae, filamentous fungi, e.g. as A. niger, and cells derived from multicellular organisms such as mammals and insects. Innummerous proteins are of great commercial interest, including from bulk enzymes, complex bio-pharmaceuticals, diagnostics and research reagents. (cf. A.M. Davy et al. 2017)