What comes to mind when you think about yeast, trusty old Saccharomyces cerevisiae? Brewers and distillers are well acquainted with yeast as the agent of fermentation. The extended use of yeast outside of its traditional areas of ethanol production and bread making has moved from the simple use of yeast extract to produce Marmite® into the areas of biomanufacturing, nutraceuticals and personal care products. The nutritional content of yeast has historically focused on yeast as a source of single cell protein and for its B vitamin content. In more recent years the market for dietary supplements has exploded and the opportunity of marketing brewers’ yeast as such has increased. Yeast based products are being proposed for use in cosmetics formulations by bulk yeast suppliers perhaps more normally associated with brewing and baking. Transgenic yeast are being exploited to synthesize secondary metabolites for pharmaceutical intermediates and mutants have been selected for the over production of fragrance molecules such as 2-phenylethanol.
Progress has been made in the exploitation of yeast as an inert carrier of small molecules. Commercial products consisting of mineral-enriched yeast have been available for some time. Zinc-enriched yeast cells are being used in the brewing industry and selenium-enriched products have been approved for use as nutrient supplements in food. The potential for encapsulating other chemicals inside yeast is less widely known. Materials that can be encapsulated in spent yeast include flavours, pesticides and active pharmaceutical ingredients (APIs).
For many years microencapsulation has been used as a tool to convert liquids to solids to improve handling. The products formed as a result of encapsulation can be stored as dry powders or granules thus volatile and flammable flavours can be handled more easily and shipped and stored with few restrictions. Microencapsulation can be used in targeted delivery to improve the impact of active ingredients, to control or delay release for masking taste and odour; to improve process stability; to protect materials sensitive to UV, heat, oxidation and high or low pH; for the isolation of reactive components and to stabilize starter cultures, probiotics and enzymes. Materials typically used include natural or synthetic polymers, gums, starches and lipids. Coatings and particles are formed using a range of methods including fluid bed coating, coacervation, spray drying and spray chilling. Typically microcapsules are formed in situ although in some cases absorption is also used to stabilize molecules, for example with cyclodextrin molecular inclusion technology or when flavours are spray dried onto maltodextrin and gums or resins.
Yeast is a natural alternative to other encapsulation materials and offers unique performance benefits. A number of encapsulation technologies using yeast have been developed and refined. The use of yeast cells as biocapsules was first considered in the 1970s when Serozym Laboratories discovered that cells (Saccharomyces cerevisiae), treated with a plasmolyzer, could be used to absorb water soluble substances for use in medical, cosmetic and food products. Yeast, widely available in large volumes as a by-product of fermentation processes, was identified as a cost effective material for use in these global industries.
Yeast has a complex structure; it comprises a dense absorbent cytoplasm rich in organelles, lipid membranes and lipid droplets, protected by a 7-12 nm lipid bilayer and heterogeneous cell wall. The surrounding cell wall is typically up to 200 nm thick, rich in beta-glucan, mannoproteins and chitin and provides robust protection to the cell contents as can be seen in Figure 1.
In 1977 the US company Swift and Co. utilized yeast strains that accumulated lipid to a high internal concentration (>40% w/w) when grown on specific nitrogen rich media. These yeast strains were particularly effective in absorbing fat soluble dyes, vitamins and drugs which dissolved in the free lipid in the cell cytoplasm. The technology, based on an aqueous mixing process, was developed further by UK based company AD2 Ltd. This was taken to a commercial scale in Europe and the USA, initially as a flavour delivery encapsulation process, by companies including Micap plc and Corn Products Corporation.
In some cases, materials can be stabilized in yeast at levels as high as 45% by weight of the powdered or granulated end product. There is potential for using waste brewery yeast or spent yeast from ethanol biofuel production with these yeast encapsulation technologies, primarily for use in food systems to improve flavour delivery, flavour stability, appearance and nutritional value. There is also potential for the encapsulation of materials for non-food applications such as pesticides, herbicides, antibacterials, antifungals and essential oils.
Traditionally excess or waste brewery yeast was sold into the malt whisky distilling industry in the UK to supplement distillers yeast and improve the flocculation properties in fermentation vessels. This was summarised in a 1985 guidance document from the Institute of Brewing and the Allied Brewery Traders’ Association. One requirement of this market is the consistent supply of viable yeast. The sale of spent Brewer’s yeast into the food processing industry has been a source of income for brewers in the past, however, the demands for higher quality ingredients at lower cost combined with the increasing costs of supply has reduced the opportunities for the brewer in this area. The supply of segregated and suitably packaged products is required to successfully penetrate the food and feed markets. Slurry, cake, dry powder and granulated products are typically available for supply. However, for food, pet food and many animal feed applications debittering to remove hop components is a prerequisite to improve palatability and may be undertaken by third party processors.
Spent yeast is typically deactivated during processing and live yeast is not required for the absorption of active ingredients to take place. The details of yeast based encapsulation using an aqueous mixing process have been described in new patents and in the scientific literature since the late 1980s.
Effective aqueous based encapsulation using yeast depends on the active ingredients to have a small degree of water solubility to facilitate partitioning of the active ingredient into the yeast inner matrix. Biocapsules typically encapsulate well those chemicals with partition coefficients around Log Po/w 2.0. A typical encapsulation process flow using yeast as an inert carrier is illustrated in Figure 2. The process can take up to 24 hours for the yeast to absorb the maximum quantity of material.
Each individual cell acts as a sink for hydrophobic molecules and can accumulate material to a high concentration. The presence of droplets found within the 4-7 micron yeast capsule, has been visualized using confocal microscopy and Figure 3 shows essential oil droplets, coloured orange, that have accumulated to around 400 g/kg dry wt.
A high degree of membrane and cell structural integrity remains following the encapsulation process. The intact cell wall and membrane can be clearly seen in Figure 5 following two hours of mixing with Tea Tree oil. Droplets a few tens of nanometers form initially and are distributed throughout the cytoplasm. These typically coalesce later in the process. Some droplets can be observed on the cell wall surface prior to being absorbed into the yeast.
The rate of absorption by spent yeast is not the same for all active ingredients and it is primarily the permeability of the yeast cell wall that acts as the selective barrier rather than the cell membrane. Figure 6 illustrates the relative potential for small molecules to be absorbed and stabilized within yeast cells. Molecules with low partition coefficients readily pass into the cells but will not accumulate and pass back out into the water during mixing. Once encapsulated, poorly water soluble materials can be dispersed more readily in aqueous systems without the aid of solvents or surfactants. The evaporation of volatile materials from the processed dry powder can be minimized thus improving shelf life performance and process stability in applications such as baking.
Molecules with low partition coefficients readily pass into the cells but will not accumulate and pass back out into the water during mixing. Once encapsulated, poorly water soluble materials can be dispersed more readily in aqueous systems without the aid of solvents or surfactants. The evaporation of volatile materials from the processed dry powder can be minimized thus improving shelf life performance and process stability in applications such as baking.
Delivering the goods
The release of components from traditional microcapsules can be achieved using physical pressure. For example, in carbonless copy paper when writing the pressure of the pen tip on the capsule containing paper breaks open the microcapsules. The encapsulated dye is released into an acid clay matrix which produces a colour change in the dye. Alternatively, in various applications, soluble starches dissolve or fat coatings melt releasing their payload. Yeast cells are robust and are resistant to physical damage, shear pressure, heat and chemical stresses. For example, dried powder formulations of food flavours can retain flavours even at 200 C. However, on the addition of water, dried yeast biocapsules are primed to release absorbed hydrophobic materials such as volatile flavour components. The release is facilitated, in most cases, by diffusion down a concentration gradient from source to sink. This happens in the mouth when food is eaten and food flavours are released into the saliva and evaporate into the buccal cavity.
Screening for pharmaceutical ingredients (APIs) from combinatorial chemistry strategies can produce 40% of targets that are poorly water-soluble. These difficult to formulate molecules are more frequently being subjected to nanotechnology approaches. There are advantages in stabilising APIs as very fine dispersions and yeast offers an alternative approach for stabilising nanodispersions by housing them within a biocapsule. Thus yeast could be presented as a vehicle for the oral and buccal delivery of these difficult to formulate APIs.
One feature of the established process is its restriction to small hydrophobic molecules and principally molecules with octanol/water partition coefficients below Log P 4.0 and molecular weights below 600 Da. It works well, for example with volatile food flavours, some small drug molecules and a limited range of crop protection active ingredients. Recent developments enable the encapsulation of large, to up to 5000 Da, and very hydrophobic compounds in biocapsules for the first time. Potential candidates include active ingredients such as insecticides (e.g. deltramethrin, ivermectin), fungicides (e.g. carboxin, epoxiconazole), molluscicides, (e.g. fentin, methiocarb), nematicides (e.g. carbofuran), rodenticides (e.g. brodifacoum, norbormide), herbicides (e.g. oxasulfuron) and poorly soluble active pharmaceutical ingredients (Class II and Class IV drugs) (e.g. fenofibrate and ketoconazole). This new approach differs from previous biocapsules processes in one key area: the removal of water from the encapsulation process. Water is replaced by a solvent such as dimethylsulphoxide in which many difficult to formulate active ingredients are readily soluble. Dilution with water after the encapsulation process can be used to stabilize the hydrophobic molecules within the cells. Following a period of mixing, the product can be spray dried; alternatively, an aqueous dispersion can be used directly. This moves us into areas where patent licensing is required, costs of processing are high and where there are demands for dedicated or GMP yeast manufacturing. This is a long way from finding an alternative use of spent brewery yeast.
Innovation using yeast biocapsules
The abundance of prior art for water based bioencapsulation systems has restricted commercial exploitation. Patents filed independently in Europe and Japan resulted in a challenging market segmentation situation. This is compounded by application based patent filings in areas such as smoking cessation products; tobacco products; homecare products; drug delivery; cosmetics; pesticides and flavouring baked goods. Many patents, particularly on the process side, have now lapsed or are due to lapse opening up the potential for commercial exploitation by the brewing and allied industries as an alternative revenue stream.
In conclusion, using spent yeast as an inert carrier may in some circumstances add value in providing a complex matrix material in which active ingredients can be stabilized as a reservoir or depot. Spent yeast is a potential vehicle to provide a sustained release profile in situ for food flavours or perhaps in the future for crop protection products. The technology is being commercially exploited already so there is clearly life in the old dog yet.
References and further reading
AD2 Ltd. European patent (1987), EP 0242135.
Bishop J., Nelson G., Lamb J. (1998). Microencapsulation in Yeast. Journal of Microencapsulation, 15, (6 ), 761-773.
Dardelle, G., Normand, V., Steenhoudt, M., Bouquerand, P.E., Chevalier, M., Baumgartner, P., 2007. Flavour-encapsulation and flavour-release performances of a commercial yeast-based delivery system. Food Hydrocolloids. 21, 953–960.
Dunlop Ltd 1986 UK Patent GB2162147
Duckham S. C., Burgess, A., Hinds, L. and Echlin, P. (2003) Microencapsulation in yeast cells - Structure and function: A cryo SEM approach. In: Proceedings of the 14th International Symposium on Microencapsulation, September 4-6, 2003, Singapore.
Kilcher, G., Delneri D., Duckham C. and Tirelli N. (2008) Probing (macro)molecular transport through cell walls. Faraday Discuss., 139, 199–212
Mattanovich, D., Sauer, M. and Gasse, B. (2014). Yeast biotechnology: teaching the old dog new tricks. Microbial Cell Factories. 13 (1) :34
Nelson G., Duckham S.C., Crothers M.E.D. (2006). Microencapsulation in yeast cells and Application in Drug Delivery. Polymeric Drug Delivery, ACS Symposium Series 923, Chapter 19, 268-281.
Normand V., Dardelle G., Bouquerand P-E, Nicolas L., Johnston D. J. (2005). Flavour Encapsulation in Yeasts: Limonene used as a Model System for Characterization of the Release Mechanism. Journal of Agricultural & Food Chemistry, 53, 7532-7542.
Many approaches have been used to encapsulate volatile flavours but one of the more unusual approaches is the incorporation of food flavours within baker’s/brewer’s yeast cells (Bishop et al. 1998). This approach has been adopted by FlavArom International Limited (http://www.flavarom.com/) and Firmenich SA (http://www.firmenich.com/) to produce stable spray dried flavour products (Normand et al. 2005). Yeast cells have a size of typically 5-7 microns in diameter and can absorb food flavours such as limonene which are stabilized internally as small droplets ranging in size from below 100 nm to around 4000 nm. Flavour loading of 20 to 40% on a dry weight basis (200-400 g/kg powder) can typically be achieved. Inclusion of nanomaterials within microcapsules, matrix particles or agglomerates that will disperse at the point of use is one means of improving the stability and handling properties of these products.
Spray drying is a common way of generating dry powders of encapsulated ingredients and particle sizes achieved are as low as 2 micron at laboratory scale or around 20-50 micron at production scale. There are now lab scale spray driers available that can produce powders with a particle size ranging from around 300 nm to 5 micron, for example the B-90 Nano Spray Dryer from BÜCHI Labortechnik AG (http://bit.ly/17OheHJ). As these systems are scaled up, the usual hazards associated with powder handling and dust in the workplace will have to be considered with a view to the challenges presented by the smaller particles that are potentially entering the atmosphere. The key is in the design and engineering controls that need to be incorporated from the outset and examples and guidance can be found from a number of sources including publications from Ostiguy et al. (2010) (http://bit.ly/1bI2Tbi) and Amoabediny et al. (2009) (http://bit.ly/1bI3fPr). A recent workshop was held by the International Life Sciences Institute and a summary report, by Howlett (2012), considered the need for providing practical guidance for the safety assessment of nanomaterials in food (http://bit.ly/17USBJe).
The production of stable emulsions with very small droplet sizes, often known as sub-micron-emulsions or nano-emulsions, is well established. Typically high-pressure homogenization is used to form lipidic bodies stabilized with an encapsulating phospholipid monolayer suspended in an aqueous environment. Alternatively, liposomes have an aqueous core enveloped by a lipid-bilayer membrane. More commonly used in cosmetics, personal care and medicinal products, they are typically unstable in most food processing environments. Ultrasound is being used to generate oil in water (O/W) and more complex double emulsions, for example water in oil in water (W/O/W), for NPD. These techniques can be used to generate stable emulsions with little surfactant with very small droplet sizes. Trials, using a rotor-stator homogenizer and ultrasonic cavitation, produced emulsions of palm oil esters with particle sizes around 63 nm (Han et al. 2012).
Many applications involve the production and use of nano-particles, nano-tubes or fibres as dispersions in liquids or as powders. Monitoring these materials has presented a number of technical challenges. There are now tools available for routinely measuring particle numbers, size, size distribution, shape and charge characteristics (zeta potential) using a variety of technologies provided by companies such as Malvern Instruments, Beckman-Coulter and Micromeritics; some companies have adopted advanced particle tracking systems to improve the quality of particle characterization, for example NanoSight Limited. However, for the very smallest of particles or droplets in the 1-10 nm range and for characterising nanostructured materials within a matrix, advanced microscopy techniques are required such as atomic force microscopy (AFM), field emission-scanning electron microscopy (FE-SEM) and cryogenic-transmission electron microscopy (cryo-TEM).
Process scale up provides a number of challenges in this area. Some companies have developed micropore membrane filtration systems, e.g. Micropore Technologies Limited (www.micropore.co.uk/). However, microfluidic technologies that could be more suitable for large scale processing typically operate at the micron scale. Scale up of nanomanufacturing is a rapidly developing field and new materials and processes are needed; we should expect to see them appearing in ever greater frequency in the near future.
Increasing the surface area by reducing particle or droplet size is one strategy for improving dissolution rates, stabilizing emulsions and improving bioavailability through increased uptake by the digestive tract. There are however some shortcomings in this approach. Increasing the surface area also increases the potential for chemical reactivity, for oxidation and other undesirable chemical interactions. Nano scale encapsulation through the use of micelles and inclusion complexation can overcome some of these limitations but will incur additional costs. Thus the potential benefits of using nanotechnology in developing novel food ingredients and products may be in niche, high value areas. This is particularly pertinent when taking into consideration the additional costs of manufacturing and process controls that may need to be implemented to ensure safe production and handling of ENMs.
There are clearly many opportunities when working with nanomaterials in the food and beverage industry particularly, in the area of natural product formulation, particularly when low fat, sugar, salt and low surfactant use are targets for novel foodstuffs development. Opportunities for modifying food structure have yet to be fully explored and the use of ENMs in beverage development continues in support of the growing use of natural colours. The influence of micellar flavour formation in aroma delivery in food and beverage systems is also the subject of a growing number of studies (e.g. Aznar et al. 2004). The greater understanding of how nanostructured materials affect the sensory impact of food and beverage ingredients will hopefully lead to new tools becoming available for NPD in the near future.
Amoabediny G, Naderi A, Malakootikhah J, Koohi MK, Mortazavi SA, Naderi M, and Rashedi H (2009). Guidelines for Safe Handling, Use and Disposal of Nanoparticles. Nanosafe 2008: International conference on safe production and use of nanomaterials. J. Phys.: Conf. Ser. 170.
Aznar M, Tsachaki M, Linforth RST, Ferreira V, Taylor AJ (2004). Headspace analysis of volatile organic compounds from ethanolic systems by direct APCI-MS. Int J Mass Spectrom; Vol. 239, (1) 17-25.
Bishop J R P, Nelson G and Lamb J (1998). Microencapsulation in yeast cells. J Microencapsul; Vol. 15, (6) 761-73.
Han NS, Basri M, Abd Rahman MB, Abd Rahman RN, Salleh AB, Ismail Z (2012). Preparation of emulsions by rotor-stator homogenizer and ultrasonic cavitation for the cosmeceutical industry. J Cosmet Sci. Vol. 63, (5) 333-44.
Higashi T, Nishimura K, Yoshimatsu A, Ikeda H, Arima K, Motoyama K, Hirayama F, Uekama K, Arima H (2009). Preparation of four types of coenzyme Q10/gamma-cyclodextrin supramolecular complexes and comparison of their pharmaceutical properties. Chem Pharm Bull; Vol. 57, (9) 965-70.
Howlett J (2012). Practical guidance for the safety assessment of nanomaterials in food. Summary report of workshop held in April 2011 in Cascais, Portugal, organised by the ILSI Europe novel foods and technology task force. pp16.
Lucas-Abellán C, Fortea I, Gabaldón JA, Núñez-Delicado E (2008). Encapsulation of quercetin and myricetin in cyclodextrins at acidic pH. J Agric Food Chem; Vol. 56, (1) 255-9.
Munin A and Edwards-Lévy F. (2011). Encapsulation of Natural Polyphenolic Compounds: a Review. Pharmaceutics Vol. 3, 793-829.
Normand V, Dardelle G, Bouquerand PE, Nicolas L, Johnston DJ (2005). Flavor encapsulation in yeasts: limonene used as a model system for characterization of the release mechanism. J Agric Food Chem; Vol. 53, (19) 7532-43.
Ostiguy C, Roberge B, Woods C, Soucy B (2010). Engineered Nanoparticles Current Knowledge about OHS Risks and Prevention Measures. Chemical Substances and Biological Agents: Studies and Research Projects IRSST Report number R-656. pp143.
Weiss J, Takhistov P and McClements D J. (2006). Functional Materials in Food Nanotechnology. J Food Sci; Vol. 71, (9) 107-16.
The use of nanomaterials in novel food and beverage applications
As with all novel developments most companies like to keep things under the radar until ready for product launch. A number of companies have at least hinted that nanotechnology plays a part in their NPD strategy. The German company Aquanova AG uses a polysorbate micelle carrier technology to solubilize a range of poorly soluble ingredients for incorporation into clear beverages. Their product range includes isoflavones, natural colours, antioxidants, vitamins and coenzymes such as CoQ10. Their technology is exemplified in the international patent WO2004002469. These micelle systems have a particle size that typically ranges from around 20 nm to 200 nm.
Creating fine dispersions of poorly soluble natural fat soluble colourings to formulate clear beverage products is one area where the technology has been applied. Casein micelles are being investigated for their ability to carry functional ingredients including Vitamin D2 and for use in clear beverages and sports drinks. International patents have been filed by the Technion Research and Development Foundation Limited, Israel; for example “Casein Micelles for Nanoencapsulation of Hydrophobic Compounds” (patent number WO2007122613).
Nanodispersions of particles of around 100 nm can remain stable for longer than conventional emulsions or dispersions and can form clear pseudo-solutions. These properties have been exploited by companies such as Food Ingredient Solutions Limited (www.foodcolor.eu) who use a protein matrix system to formulate natural colours such as astaxanthin for use in beverages. Compass Foods have developed Habo Monoester P90, a monopalmitate sucrose ester surfactant to prepare micellar dispersions of 20-80 nm particles (http://bit.ly/1emuNNV). They propose the option of using nanocapsules containing antioxidants to stabilize the co-encapsulated natural colours. Incorporating colours into nano- and microcapsules to stabilise them may have an impact on their light scattering properties, as the phenomena of Rayleigh scattering and Mie scattering come into play. This means that additional development work may be required to achieve the desired colour outcome. A number of companies have addressed the challenges of formulating natural flavours and are regularly demonstrating their innovative products at the various global ingredients business exhibitions.
Cyclodextrins have been successfully used to encapsulate a wide range of fat soluble functional ingredients by forming molecular inclusion complexes. Cyclodextrins are a form of modified starch with a ring structure made up of glucose units. The most common ones in routine use comprise 6, 7 or 8 member rings and are termed alpha- beta- and gamma-cyclodextrin (figure 2). They have a small outer diameter of less than 2 nm. These molecules have a polar outer surface allowing them to dissolve quite well in water. Their inner core forms a non-polar cavity into which can fit small fat soluble molecules such as many food flavours, fat soluble vitamins and natural colours such as carotenoids. This enables them to be used to help to solubilise poorly soluble ingredients. The three main forms are now being used more widely in new product development (NPD) as approval for their use as a novel food ingredient proceeds through the US, Japan and Europe.
Figure 2. Simplified diagram of the chemical structure of alpha-, beta- and gamma cyclodextrin.
Cyclodextrins have been utilized by the UK company FlavorActiV Limited for over ten years to stabilise flavours, off-flavours and taints. The resulting powders are used for training sensory panels to recognise positive and negative attributes in products, for improved quality management and process control, in the brewing industry and in the wider food, beverage and water industries.
Cyclodextrins are being adopted in the pharmaceutical industry to improve the delivery and bioactivity of active ingredients and are also being used in food supplements and can be found for example in formulations of CoQ10. Tishcon Corporation in the US has commercialized this type of product with their HydroQSorb® product (http://bit.ly/19qpDLB). The performance of a number of different formulations of complexed CoQ10 has been reported by Higashi et al. (2009).
Cyclodextrins have also been investigated for a number of other applications, such as improving the stability of bitter tasting products containing compounds including flavonols (e.g. quercetin, and myricetin) by Lucas-Abellán et al. (2008); taste masking of ginseng and green tea (Wacker AG information sheet “Masking tastes and odors with CAVAMAX® cyclodextrins” (http://bit.ly/1acC16q). A more general review of polyphenol encapsulation has been recently published by Munin and Edwards-Lévy (2011) that details the range of techniques used to encapsulate these often unstable and unpalatable functional ingredients.
In Part 3 we consider what we might mean by the term "nanostructured materials" and is also where you can find further reading and a list of references.
Craig is a consultant, a flavour enthusiast and an unapologetic analytical chemistry geek.