Fermentation offers limitless supply

The range of fermented ingredients is limited only by the imagination and physical laws of nature (and operating cost). From renewable sources of nutrition to sustainable biofuels, the future may be fermented. Jon M Hansen and Joe Pfeifer give an overview of the technology's promise

Martek's algal DHA fermentation research facilitiesFermentation is an anaerobic or aerobic process that utilises the cultivation of micro-organisms to produce products metabolically. Biochemically, fermentation is an anaerobic process, whereas industrial fermentations generally refer to both aerobic and anaerobic processes.

The use of anaerobic fermentation has existed for thousands of years starting with the production of beer in ancient Egypt and fermented foods in India. Some of the earliest industrial fermentations were for the production of penicillin during World War II. Today, physical conditions such as temperature, pH, pressure and dissolved oxygen are typically monitored and controlled in industrial fermentations. Other variables are often monitored or controlled, depending on the sensitivities of the organism and process. The fermentation conditions vary depending on the organism and product of interest.

Fermentation products can be broken down into several basic categories. These include but are not limited to primary metabolites, secondary metabolites, energy storage, enzymes and whole-cell technologies. A wide variety of commercial products are made from fermentation for use in industrial, food/beverage, pharmaceutical, nutritional-supplements, biofuels, cosmetic and animal-feed applications.

Industrial products include small molecules such as amino acids, organic acids (eg, lysine, acetic acid, citric acid), vitamins, and other short-chain organic compounds that have a wide range of commercial applications. Medium-size molecules, such as fatty acids, isoprenoid compounds and proteins/enzymes, are also made by fermentation. There are a number of large molecules such as biopolymers (eg, bioplastics, polysaccharides) that are made by fermentation. Intact whole-cell technologies include single-cell proteins such as animal feed and yeast extract, single-cell oils (such as Martek products), and probiotics. Furthermore, improved lot-to-lot consistency, fewer impurities such as pesticides and contaminants, and reduced cost are also possible from an optimized culture and fermentation process designed for specific products.

Fermentation development typically starts out using a wild-type organism or an organism that has been through classical strain improvement. This is often coupled with a bio-rational strain selection approach and/or high-throughput screening technologies. Genetically modified organisms may also be developed in an effort to achieve higher product yields, or to manufacture products that cannot be produced cost effectively by other means. The desired product either accumulates within the cell or is secreted into the fermentation broth. For certain applications, the intact cells of the microorganism are the desired product.

Large-scale industrial fermentation technology has been around for nearly 80 years. A typical industrial fermentor consists of a large cylindrical tank with a height of four to six times the tank diameter. The tank is mixed through the use of a mechanical agitator and/or a compressed-air supply. Fermentation inlet air is usually dried and filtered. Nutrients and water added to the fermentor are typically sterilised using steam or filtration. Fermentation exhaust air is usually condensed and/or filtered. The majority of industrial fermentations are done under hygienic or aseptic conditions to minimise the growth of contaminants. To facilitate this, the systems are usually constructed using stainless steel to minimise corrosion and equipment deterioration.

There are several different fermentation cultivation methods. Most cultivation processes are either batch, fed-batch or continuous. In a batch process, all of the required ingredients are added to the fermentation at the beginning of the process, along with the addition of the micro-organism. In a fed-batch process, the organism is fed one or more nutrients, such as carbon and/or nitrogen, during a portion of the fermentation, thus enabling control of these variables at an optimum level throughout the process. In a continuous fermentation process, nutrients are added and cells/product(s) are harvested in a continuous manner to maximise product output and minimise vessel downtime.

On-line and at-line fermentation monitoring has progressed from the basics of temperature, pH and dissolved oxygen to more advanced measurement and detection methods. These can include off-gas mass spectrometry, Fourier transform near infrared (FTNIR), HPLC, online enzymatic analysis, dissolved carbon dioxide, various online electrolytic measurements (sodium, potassium, ammonium, etc.), fluorescence-based detection of various compounds, viable cell sensors, vessel liquid, and foam levels. A wide array of off-line analyses is also employed depending on the culture, process and product.

With advancements in process automation and information technology, the capability to analyse large amounts of fermentation data to optimise processes is greatly enhanced. Improvements in instrumentation and control technologies, in addition to batch automation capabilities, have enabled further advances in fermentation technology.

Recently, improvements in genetic modeling, sequencing and characterisation have provided additional tools that could prove to have a significant effect on future discovery and development, especially when coupled with fermentation, downstream processing and strain-improvement efforts. Computational fluid dynamics and other modeling tools can provide a greater understanding of scale-up and lead to improved fermentor design.

The future of fermentation holds many different directions that are limited only by the imagination and physical laws of nature (and operating cost). Some examples of cost-effective future industrial fermentation may include meal-replacement technologies, various renewable sources of food/nutrition, cosmetics, industrial products that are currently petrochemically based, novel biofuels and any number of recently identified bioactive compounds.

Fermentation offers an environmentally sustainable, contaminant-free production alternative to current methods of harvesting these products that may otherwise deplete our natural resources and harm our environment.

Jon M Hansen and Joe Pfeifer are directors of fermentation sciences at Martek Biosciences.

With fermentation, all things are possible

Fi talks fermentation with Bob Hutkins — Nutracon '09 featured speaker and professor of food science at the University of Nebraska — in particular about the notable concept of probiotic bacteria's ability to ferment prebiotic fibres.

Fi: What advantages does fermentation offer over conventional sources of nutritious ingredients? Longer shelf life? Consistency? Taste?

BH: In ancient times, fermentation was one of the main ways foods could be preserved, and even though preservation is still an important property, there are now many other ways for preserving foods. However, fermented foods have other important advantages. For one thing, they have sensory properties — flavour, aroma, appearance and texture — that are often dramatically different from the starting material (think Roquefort cheese compared to milk).

Fermentation also enhances the functionality of otherwise ordinary ingredients; thus, adding the right micro-organisms to a simple flour-water dough not only makes possible the creation of a crusty baguette, but fermentative sourdough organisms can be used to make rye and other flours more amenable to breadmaking. Likewise, the unique flavours and intoxicating properties of wine, beer and other alcoholic properties can only be achieved by fermentation.

Fi: What about enhanced nutrition?

BH: Fermented foods can also serve as an excellent source of nutrients. Obviously, fermented foods such as cheese, yoghurt and sausage provide protein, minerals and vitamins, but there are also many important micronutrients that fermented foods provide. For example, wine contains several polyphenols, such as resveratrol and flavonoids, that are thought to reduce cardiovascular disease. Tempeh, a fermented-soybean product that originated in Indonesia, contains vitamin B12, which is important for vegetarians. So while one can obtain resveratrol or B12 from a pill, it seems to me that the best way to deliver these nutrients is naturally via fermented food.

Finally, many fermented foods contain live micro-organisms that may survive digestion and reach the intestinal tract where they can then contribute to intestinal health. Yoghurt, of course, is the best-known example of a food that serves as a vehicle for health-promoting bacteria. But many fermented foods, provided they aren't heat-treated after fermentation, may serve a similar role. Thus, fermented vegetables, such as unheated sauerkraut or its Asian counterpart, kimchi, contain billions of lactobacilli and other bacteria per serving, and even some fermented sausages, such as the dry, uncooked versions one finds in Europe, also contain live lactic-acid bacteria.

Fi: You've done interesting work on lactic-acid bacteria and bifidobacteria's ability to ferment probiotic FOS. Is the synbiotic concept for real?

BH: Our research is focused on factors that influence the ability of probiotic bacteria to persist in the intestinal tract. A common misperception is that if one consumes a so-called probiotic organism, that it always survives digestion and reaches the colon in a live and viable form. Unfortunately, this is not necessarily the case, and even if the organism does manage to survive the trip, there is no guarantee that it will be able to compete successfully with the trillions of bacteria that already reside there. Instead, the newcomer may simply wash right on through without having enough time to become established or to perform health benefits.

If, on the other hand, that probiotic organism was provided with a nutrient that it alone (or almost alone) could utilise and that its competitors were unable to efficiently use, then the probiotic would stand a much better chance of persisting in that environment. This scenario describes the role of prebiotics — nutrients that selectively stimulate growth or activity of favourable bacteria including probiotics.

Our work has shown that metabolism of prebiotics by probiotic bacteria is not a universal trait. Rather, the genes encoding for the relevant metabolic pathways are present in some organisms and absent in others. There also is variation between different strains of the same species with regard to the route by which a prebiotic is metabolised. Differences in how different strains ferment prebiotics may also have implications with regard to how effective the prebiotic ultimately will be.

Fi: This is one of the more important applied-research questions: which probiotic strains best match up with which prebiotic fibres?

BH: There are an array of prebiotics in the marketplace, ranging from the short-chain fructo-oligosaccharides and galacto-oligosaccharides (GOS), to the longer-chain polysaccharides such as inulin and resistant starch. We have shown, for example, that some organisms are equipped to ferment some prebiotics but not others.

In one study from my lab, we showed that a particular strain of Lactobacillus paracasei grew very well on FOS and inulin, but poorly on GOS, whereas a strain of Lactobacillus acidophilus grew well on GOS but not at all on FOS or inulin. Although these data may appear to be somewhat confusing, this really illustrates the metabolic diversity that exists among these bacteria and highlights why conducting these sorts of experiments is so important.

Ultimately, this work speaks to the issue of compatibility — that is, if manufacturers formulate a food or supplement to contain both probiotics and prebiotics — a synbiotic — and they expect the prebiotic to stimulate that probiotic, then there should be experimental evidence to show that the probiotic can indeed ferment that prebiotic.

Fi: Prebiotics seem to be riding the coattails of probiotics. Do prebiotics demonstrate efficacy alone?

BH: It is worth noting that while the synbiotic approach has considerable merit, prebiotics by themselves are capable of stimulating members of one's own microbiota, and there may be advantages with that approach as well. In other words, individuals may already harbour bifidobacteria and lactobacilli in their GI tract that will respond to prebiotics.

In addition, prebiotics may also have an altogether separate benefit, by virtue of their ability to inhibit pathogenic bacteria from adhering to intestinal cells.

Fi: What do you think is the outlook for probiotics?

BH: Obviously, from the food side, there are more and more probiotic strains being introduced into a wide assortment of foods. Although probiotics must meet a number of important criteria — high numbers, viability, safety, survival through the digestive system — the main scientific issue for probiotics, in my opinion, is validating that the strain provides a clinically proven health benefit.

This last criteria is the most difficult to satisfy, since it requires testing using human subjects. A lot of strains have good in vitro data and can be packaged to deliver high numbers of viable cells, but human feeding studies, with the outcomes published in scientific journals, are the ultimate test. Nonetheless — and this shows how much the field has advanced in recent years — there are now quite a few strains and probiotic products for which reasonable data exist to support health claims.

Fi: Has the market moved ahead of the science — that is, everyone wants probiotics integrated into foods, especially with health claims stated or suggested on the label, but the research has yet to definitely validate specific health effects?

BH: The development that continues to have a huge impact on the field relates more to the science and the new technologies that have been developed to study probiotics and prebiotics. There are trillions of bacteria that live in the human intestinal tract, most of which can't be grown in the laboratory. It is now possible, using high-throughput DNA sequencing technologies, for scientists to identify most of those bacteria and to assess the changes that occur when subjects consume pro- or prebiotics, or simply change diets in other ways.

There are also sophisticated analytical tools that can be used to detect metabolic changes or the appearance of disease biomarkers or immune system effectors that form under those same conditions. These new tools and technologies will not only influence the probiotics field, but, now that the intestinal microbiota has been shown to affect not only intestinal diseases but also obesity, diabetes and other systemic diseases, they also have the potential to have a much broader impact on our understanding of health and disease.

Fi: Is it fair to say that not all pro-biotics are created equal?

BH: It is important to note that health benefits are strain-specific. That is, while a given strain may be effective, for example, at reducing IBS symptoms, that same strain may have no impact on other health issues. So while there are numerous strains in the probiotics marketplace, it may well be that those strains that can 'prove' themselves with documented health benefits will be the ones that become accepted by the medical community and ultimately by consumers.

Select suppliers: providing a broad range of fermented ingredients

Embria Health Sciences
Embria Health Sciences' key ingredients are the result of its proprietary fermentation and drying technology. Based on a multistage process that has set a standard in the animal-nutrition industry for more than 60 years, this process produces unique metabolites that provide beneficial nourishment and superior bioavailability. Flagship ingredients include eXselen high-selenium yeast and EpiCor, a high-metabolite immunogen.

Draco Natural Products
Draco offers fermented botanical products used in TCM, such as red yeast rice extract from Monascus purpureus yeast and Cordyceps sinensis extract from Paecilomyces hepiali fungus. Massa fermentata is used in TCM for indigestion, loss of appetite and diarrhea. Yeast extract, a water-soluble/dispersible product from Saccharomyces cerevesai and a rich source of beta-glucans and growth factors, has use in immune-support products and cosmetic applications. Draco's custom fermentation of botanicals can be used in drinks, supplements, cosmetics, and functional foods. Organic and Kosher certifications of its fermentation production line will be completed by spring.

Fluxome is a leader in the field of industrial biotech. Its patented technology allows it to produce nutraceutical ingredients via fermentation of baker's yeast. Fluxome uses its microbial metabolic-engineering technology platform to build efficient cell factories to produce a broad range of nutraceutical ingredients. In 2008, Fluxome and CP Kelco began jointly developing and commercialising fermentation-derived resveratrol for the supplements, foods, beverages and cosmetics markets.

Kaneka, the world's largest co-Q10 manufacturer, has been manufacturing natural co-Q10 using the yeast fermentation method for more than 30 years. Commercial co-Q10 is manufactured by three different processes: the yeast fermentation method, a fermentation process using bacteria, and a synthetic method that uses solanesol derived from tobacco as starting material. KanekaQ10 is the world's only yeast-fermented co-Q10, bio-identical to the co-Q10 produced within the body.

Martek Biosciences
Martek Biosciences is the world's largest producer of fermentation-based, omega-3 vegetarian oils. Its production facilities have the capacity to produce several million litres of fermentation. Martek produces life'sDHA, a sustainable and vegetarian source of the omega-3 fatty acid DHA (docosahexaenoic acid), for use in foods, beverages, infant formula and supplements. The company also produces life'sARA (arachidonic acid), an omega-6 fatty acid, from a sustainable, vegetarian source, for use in infant formula.

Nutraceutix operates two facilities. One specialises in fermentation and raw-materials preparation — drying, blending, packaging — and the other in cGMP manufacturing and private labelling of finished product capsules and/or tablets. The fermentation facility produces more than two dozen probiotic species for supplements, food additives and feed additives, and bacteria for its proprietary Live-Bac Tableting Process that offers improved shelf life for Lactobacillus acidophilus supplements. L acidophilus, L salivarius, L plantarum and Bifidobacterium bifidum are some of its best-selling strains.

Solazyme Health Sciences
Solazyme Health Sciences is the Health and Wellness division of Solazyme Inc, a global leader in algal biotechnology and a provider of a wide array of sustainable product technologies. Based in California, the company offers a portfolio of ingredient and product innovations for licensing to marketing partners in the nutraceuticals, functional-foods and cosmetics industries. Solazyme is devoted to harnessing the energy-harvesting machinery of various species of algae to produce valuable products via fermentation.

Toyo Bio-Pharma
Toyo Bio-Pharma's fermented extracts undergo a cutting-edge manufacturing process, resulting in powdered final products that are versatile for use on their own or as key ingredients in manufacturers' proprietary blends. The company utilises its fermentation technology with fruits and vegetables such as cabbage, onions and carrots, which enhances the bioavailability of the active compounds found in fruits and vegetables, making them more potent, aromatic and flavourful.

ZMC-USA produces fermented co-Q10. A cold-water dispersible powder at varying concentrations can be used for fortification of water-based foods, instant products, puddings, confectionery, milk products and effervescent tablets. It is also available as tablet-grade powder for tablets and hard-shell capsules, and crystalloid for soft capsules. A benchmark study was conducted on its co-Q10 by two independent labs in Japan. The assay consistently averaged 99.69 per cent purity. The cis isomer content, which is found in synthetic material, is nonexistent. This benchmark confirms no unknown impurities exist in ZMC's co-Q10.

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