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Simple sugars aid preservation of bacterial probiotics

Juan J. de Pablo

Juan J. de Pablo view
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Simple sugars that naturally help brine shrimp and other organisms survive lengthy dehydration form the core of a preservation technique developed by Chemical and Biological Engineering Howard Curler Disinguished Professor Juan de Pablo. Incubation in a defined mixture of sugars and other compounds could potentially protect a host of biological molecules, bacteria and more complex cells during freezing, drying, storage, and re-wetting, de Pablo found. But the technique's first proven use has been to preserve bacteria sold as probiotics — microorganisms that when consumed confer health benefits such as boosting the immune system and aiding digestion. Rhodia, Inc. of Madison, Wisconsin, holds an exclusive license to de Pablo's advance from the Wisconsin Alumni Research Foundation (WARF). The company has developed the technology into a versatile system for extending the room temperature shelf-life of freeze-dried Lactobacillus acidophilus, its top-selling probiotic product.

"When you sell dry powders of probiotic bacteria as dietary supplements, maintaining bacterial viability over months or years is a key challenge," says Rhodia scientist and technical manager of probiotics, Greg Leyer. To ensure probiotics were alive when purchased by customers, Rhodia's products in the past required refrigeration, which limited their market.

"Unlike dairy products that people are used to keeping cold, stocking probiotics in the refrigerator case at the pharmacy or grocery store really limits who is going to see or buy the product," says Rhodia's director of business development, Scott Bush. "So, a major objective of ours was to take these products out of the refrigerator and maintain their stability on the shelf."

Enhanced by Rhodia during the commercialization process, de Pablo's technique now allows the company to revive more than 70 percent of the original L. acidophilus cells in a culture after freeze-drying and storage for 18 months at room temperature. Previously, only about 5 percent of cells survived under the same conditions.

"Not only will the new system allow us to grow our product sales in the health market," says Bush, "but we're also commercializing this technology to stabilize probiotic bacteria in infant and toddler formulas, instant breakfast drinks, dairy products and a number of other applications as we speak."

Rhodia first approached de Pablo when the professor was just beginning to investigate certain sugars, known as disaccharides, as preservation agents. The idea to use disaccharides comes from nature, says de Pablo. For example, powdered brine shrimp eggs contain large amounts of the disaccharide trehalose. And the seeds and embryos of plants, which must survive dehydration during seed dispersal, contain high concentrations of sucrose.

Although de Pablo had already used his technique to increase the activity of an enzyme by as much as five-fold after drying, the task that interested Rhodia was much more complex.

"When you freeze and dry an enzyme, all you need to preserve is the molecule's structure," says de Pablo. "But when you freeze and dry a bacterium, you need to preserve life, the integrity of the entire cell." Working closely with Rhodia scientists, de Pablo and his graduate students conducted a series of experiments in which they incubated L. acidophilus in concentrated solutions of single disaccharides, including trehalose, sucrose and maltose, prior to freeze-drying. They then tested the microbe's ability to recover after weeks or months of storage at temperatures as high as 98 degrees Fahrenheit.

To increase the sugars' stabilizing effects, the researchers added borate or phosphate to the mix. These ions, which create linkages between the sugar molecules, increased the survival of L. acidophilus cells by more than 20 percent after being freeze-dried and held for several months at high temperature.

One way disaccharides help to preserve cells is to inhibit the formation of ice crystals, which can damage cellular contents during freezing. The sugars cause frozen water to take on a less destructive, amorphous structure that, although solid and mechanically stable, lacks the ordered arrangement of a crystal. The addition of phosphate increases this effect.

Disaccharides also help maintain the integrity of cell membranes when bacteria are frozen, dried and rehydrated; otherwise cells tend to become leaky and spill their contents during these processes. Other mechanisms of protection are areas of active research in de Pablo's laboratory.

Beyond the solution of Rhodia's industrial problem and expansion of de Pablo's research program, the collaboration has produced other rewards. His graduate students benefited greatly from their interactions with Rhodia scientists and engineers, de Pablo says, particularly during the period when the company scaled-up his bench-top system to the commercial level.

Rhodia for its part found that de Pablo's basic research perspective complemented its more practical approach to problem-solving. "Being a commercial laboratory, we often don't have the time or resources to ask why something works," says Leyer. "But it's important for us to try to understand why so that we can develop new, improved stabilization formulations with some basis of understanding. So, we appreciated the academic part of Juan's work."