Insights into Protein Analysis, from Commonly Used Methods to New Developments

Posted on:November 21, 2014
Protein separation--factors and considerations

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November 21, 2014, Global Food Forums — The following is an excerpt from the Arla Foods’-sponsored “2014 Protein Trends & Technology Seminar Report: Formulating with Proteins.”  

Protein is vast and complicated and, as such, needs equally complex methodologies to analyze it. Joe Katzenmeyer, Chemistry Supervisor of rtech Labs, provided a crash course on the subject.

“I like to tell people that the work my lab does bridges the gap between very traditional technology and very modern technology,” Katzenmeyer said, adding that his lab analyzes between 1,500-2,000 protein samples per month.

On the traditional side, there’s the Kjeldahl Method, which has evolved quite a bit since 1883 and is still widely used. In it, samples are boiled in sulfuric acid to convert the nitrogen into ammonium sulfate. The distillation involves adding sodium hydroxide and steam heat to release the nitrogen as ammonia; then the ammonia is captured in a boric acid solution that is titrated with hydrochloric acid.

“The majority of our work is done by this method. In a good day, we have two shifts that can run about 10 racks of samples,” Katzenmeyer said. “It’s our most high-throughput
method and the most commonly used for nutritional labeling.”

The Dumas Method is another traditional technique for protein analysis that involves the combustion of the sample and collection of the resulting gases. It involves far fewer chemicals and far less time than Kjeldahl, which is increasing its popularity, Katzenmeyer said. It involves applying high heat in a pure oxygen atmosphere; collecting gases; removing carbon dioxide and water; and analyzing the remaining nitrogen with a thermal-conductivity detector.

Both the Dumas and Kjeldahl methods are highly precise, with easily comparable results, but they have some obvious drawbacks. Non-protein nitrogen can be interpreted as protein, so one doesn’t know which proteins are involved, and one needs a multiplication factor to turn the nitrogen content data into protein.

More modern methodologies rectify those issues, and separation systems have been developed to determine the presence and amount of non-protein nitrogens
and individual proteins, such as myoglobin, denatured/undenatured whey, casein and alpha-lactalbumin.

Gel electrophoresis, for instance, uses electricity to separate and quantify protein macromolecules and fragments based on their size and charge. Liquid chromatography is another example, which takes liquid proteins (often dairy) and passes them through a column for the same purpose.

There is also mass spectrometry, which ionizes the components of a sample with an electron beam and then separates them with electromagnetic fields.

Of the more recent methods, however, near-infrared reflectance is most on the rise because of its incredible speed. It uses an infrared light directed at the sample, which causes vibrations in the chemical bonds and creates an energy spectrum that is analyzed and calibrated. “You could have a protein content reading on the production line in, say, 30 seconds,” Katzenmeyer added.

These recently developed methods, while more informative and specific, are generally more costly to execute. “They don’t separate very well, which is the difficulty with proteins. Imagine you have 10,000 proteins—you can’t fit all those peaks [one for each protein] in one little window. So, it gets much more complicated; more labor-intensive; and more expensive,” Katzenmeyer said.

Joseph Katzenmeyer, Chemistry Supervisor of rtech Laboratories,,  651-375-2207



A Food Scientist’s Approach to Working with Organics

Posted on:

November 21, 2014, Global Food Forums — The following is an excerpt from the Ingredion-sponsored “2013 Clean Label Conference Report.” 

Herzog-organic-clean label-Global Food Forums

The process of developing organic products is heavily influenced by the percent of organic components in the final product; additional certifications required; any retail requirements; and/or internal company requirements. Photo courtesy: (C) iStockphoto

Organic consumers show a wide spectrum of behaviors, said Sharon Herzog, Director of R&D, Country Choice Organics. One category, which comprises less than 10% of American organic buyers, is the “true natural:” those with a “faith-based belief system” and who are “committed to organic and prioritizes health and environment over price, convenience or taste.”

A second type, composed of the “health seeker,” encompasses approximately 20-25% of households. These consumers are “faith-based decision makers” who are “committed to personal/family well-being, but are not willing to sacrifice taste or convenience for a health benefit.”

Some of the most prominent drivers of the organic movement include consumer awareness of the link between nutrition and health, and the desire to avoid pesticides, herbicides, GMOs and trans fats. Further drivers include concerns for the environment and an interest in sustainability.

Her unique Product Development Toolbox for organic products addressed regulatory compliance, knowledge of ingredients, and their functionality processing and
packaging. The process of developing organic products is heavily influenced by the percent of organic components in the final product; additional certifications required; any retail requirements; and/or internal company requirements.

For example, which ingredients can be used and what claims can be made depends on whether the finished product contains 100%, 95% or more, at least 70%, or less than 70% organic material in the final product (not counting its water and salt content). Please see Permitted ingredients are also determined by the National List of Allowed and Prohibited Substances (which can change rapidly and for which there is a Sunset Process—all ingredients are reviewed at least every five years); the availability of a non-organic ingredient declaration; an ingredient’s commercial availability; and certain other certification requirements.

Some non-organic, agricultural substances are allowed, because they are not commercially available. Herzog discussed the challenges surrounding ingredient functionality by using emulsifiers as one example. For the conventional emulsifiers mono- and di-glycerides, organic substitutions could be lecithin, rice bran or oat fiber.

When it comes to lecithin, the form is also important. The liquid form must be organic, since it is commercially available. However, non-organic, de-oiled, powdered lecithin is allowed for use in certain organic products—since this form is not considered commercial availability. When a humectant or moisture control is needed, HFCS is a conventional choice, said Herzog. Organic replacements might be brown rice, cane, tapioca or oat syrups.

There are considerations in product scale-up with organic ingredients. For example, organic sugar generally has not had all molasses removed, and clumping can be an issue. In regards to antioxidants, conventional choices include TBHQ/BHA, whereas alternatives for organic products could be tocopherols and/or use of ascorbic acid, nitrogen, high-oleic oils and cinnamon.

Turning to flavors, Herzog noted that natural flavorings can be used, but one must dig deeper than that for their use in organic products. For example, carriers in a flavoring
cannot be synthetics (e.g., propylene glycol, polyglycerol esters of fatty acids, mono- and di-glycerides or polysorbate 80); and no synthetic preservatives are allowed (benzoic acid, BHT/BHA). During its processing, certain solvents are allowed (e.g., water, natural ethanol, super-critical CO2, essential oils, natural vegetable oils), but not hydrocarbon solvents.

Herzog ended her presentation by noting that at her first natural products show, an organic product retailer said “Sharon, we’ll never have to apologize for what we do” and noted that she does feel really good about the industry.

Sharon Herzog, Director of R&D, Country Choice Organics,


Speakers at Global Food Forums Events

Posted on:November 20, 2014

Challenges and Solutions When Working with Protein and Fiber

Posted on:November 13, 2014

 November 13, 2014, Global Food Forums — The following is an excerpt from the Arla Foods’-sponsored 

The challenges of fortifying with protein & fiber, Martha Porter, 2014 Protein Trends & Technology Seminar“2014 Protein Trends & Technology Seminar Report: Formulating with Proteins”  

Protein and fiber are added to food systems for many reasons, both functional and nutritional. However, with their addition comes the need for ingredient and processing adjustments, depending on the final food and its desired characteristics.

“The approach that works for us,” explained Martha Porter of Merlin Development, “is to first identify all issues through searches of literature, marketplace, patents, competitive
products (both retail and restaurant) and analogous foods. Then robust experimental design optimizes taste, cost, process and shelflife. Finally, confirmation runs verify the design predictions.”

Porter went on to highlight key considerations when using protein in low-, intermediate- and high-moisture systems. Low-water Systems: In low-water systems, such as protein bars, texture changes over shelflife. Protein tends to increase firming over time, beyond the normal firming that takes place. Proteins are not fully hydrated immediately
and, over time, they draw moisture from syrups generally used to hold bars together. Fiber, if it is not fully hydrated, can also draw moisture from the syrup. The continuous syrup phase then becomes more concentrated, contributing to the loss of pliability.

“Strategies to overcome these issues include use of multiple sources of protein and fiber,” said Porter. In addition to protein powders, nuggets or crisps can be high in protein and also contain fiber. Coatings can be protein- or fiber-fortified.

Cereal pieces, like oats, wheat flakes, nuts, pulse flour, or pieces and seeds, are other sources of protein and fiber. Protein hydrolysates are helpful to mitigate firming. Low-DE syrups promote chewiness and help maintain pliability. They contain longer-chain carbohydrates that hold onto water better and provide cohesiveness.

Higher-DE syrups add sweetness; multiple forms of sugar (sucrose, fructose, etc.) in the same binder system can hinder recrystallization. Sugar alcohols control water activity
and browning. Typically, granola or cereal bars need a water activity below 0.65, with pH in the acidic range. Intermediate-water Systems: Intermediate-water systems like bread have sufficient water to hydrate ingredients, such as fiber and protein, but there is limited
room for their fortification due to other necessary functional components. For example, dilution of gluten creates problems with volume and texture in bread.

In bread, protein considerations include clean flavor and color, especially in white bread. Non-white breads can incorporate pigmented particulates, like nuts, seeds and other
whole-grain ingredients. Fibers can include resistant starches and maltodextrins, which are digestion-resistant, but behave like starches and maltodextrin. They can help mitigate the heavy texture seen with high-cellulosic fiber breads. A blend of different fiber sources may be necessary to achieve both nutrient content and organoleptic quality. Other formula and processing adjustments may be necessary as well, said Porter.

High-water Systems: In beverages, protein selection depends on the desired characteristics of the final product. If clarity is desired, acidified proteins are needed. The proteins used will also depend on the desired function of the beverage or nutrition claims.
Ionic strength, pH, fat and carbohydrate content, and processing parameters, such as temperature and shear, affect final product characteristics. For fiber, the focus is on nutrition, but beverages need fibers with a minimal impact on viscosity, explained Porter.

High-protein and -fiber solutions can be gritty, which can be masked by viscosity. Soluble
fibers may be more helpful, as can smaller particle size. Processing parameters in beverages that need consideration include rehydration time; heat stability of the protein;
and turbidity after heat treatment and fiber dispersibility. Homogenization and emulsion formation, batching temperatures, order of ingredient addition (critical for an acidification step) and packaging (clear or opaque) also help determine final product qualities.

In summary, determination of the rationale behind product fortification is first and foremost. Different moisture levels determine how to approach the formulation issues. Protein and fiber selection can be critical to product success. Process considerations also matter.

Martha Porter, Scientist, Merlin Development Inc., 763-475-0224,,

Taste Physiology and Considerations in Sweetener Choices

Posted on:November 12, 2014

November 12, 2014, Global Food Forums — The following is an excerpt from the Ingredion-sponsored “2013 Clean Label Conference Report.”

Alex Woo discusses sweetness enhancement and how the brain processes taste.

Cross-modal correspondence can enhance sweetness. The brain processes information from different
senses to form multisensory experiences. For example, smells, other tastes, trigeminal sensations, sights (like colors) and sound all influence taste.”

When it comes to making foods sweeter in a “clean label way,” there are ways to do it naturally and simply, besides using sugar. Some approaches take advantage of the connection between taste and smell. The trigeminal nerve is found in the face (rather than the nose). It responds to irritants, like tingling and numbing, as well as temperature differences.

“The trigeminal sensation can also be used for sweetness enhancement, as can all of the other senses,” said Alex Woo, Managing Director of W2O Food Innovation, as he discussed recent technologies in clean labeling sweetness enhancement.

Natural high-potency sweeteners, such as stevia and monk fruit extract, offer solutions in reduced-sugar or sugar-free applications. When using these sweeteners, a bulk sweetener is also sometimes needed, such as natural non-/low-caloric erythritol, which helps achieve maximum sweetness, yet with minimal off-flavors and low cost, suggested Woo.

Stevia extract, which is labeled as such, is commonly used and has multiple suppliers. It is “natural,” non-caloric, has GRAS status with no FDA objection letter, is 200-400 times sweeter than sugar, stable to heat and a pH over 3, is non-GMO; and certifications for kosher and halal are available. Monk Fruit extract is also non-caloric and is GRAS with no FDA objection letter. It is not yet approved in the EU. Monk fruit extract, not quite as common yet, is 150-200 times sweeter than sugar, heat-stable, non-GMO, kosher-certified and is labeled as “monk fruit extract.”

“Monatin” is a unique, natural amino acid that has recently emerged, but, as yet, is not approved anywhere. It is extracted from a South African plant, Sclerochiton ilicifolius root. It is 3,000 times sweeter than sugar with a unique temporal profile. Monatin has a quick sweetness on-set and no lingering, bitter, metallic or astringent aftertaste.

Woo went on to explain that erythritol has multiple suppliers, is found in fruits and vegetables, and is the only atural polyol made by fermentation. It also has the highest
digestive tolerance among all polyols. Non-caloric, it is non-GMO-possible, 65% as sweet as sugar and has a 3.5% limit in beverages in the U.S. However, not all consider erythritol a clean label solution.

“When ‘natural’ is not enough,” Woo gave examples for sweetener enhancement that could result in shorter label declarations. He explained how to use “cross-modal
correspondences” to enhance sweetness. The brain processes information from different senses to form multisensory experiences in people’s daily lives; therefore, smell, tastes other than sweetness, sights, sounds and trigeminal sensations can all influence the perception of sweetness.

Although sweetness is detected in the mouth, there is also interaction between olfaction and gustation. Retronasal “sweet” aromas sensed in the nose increase the sweet perception in the mouth. Many sweet taste modulators are legally labeled as “natural flavors,” thus result in more consumer-friendly labels.

Woo referenced work by Professor Tepper at Rutgers University, who is investigating molecular biology as a way to “trick” the taste buds. “This approach is novel in the food industry,” stated Woo, “but it is the way of the future.” For example, fresh tomato aroma makes tomato sauce taste sweeter. Sugar distillates enhance beverage sweetness.

Vanilla, below and above threshold, enhances sweetness, according to various reports. Some FDA GRAS, natural, high-potency sweeteners are approved under FEMA GRAS as “natural flavor,” when they are used at very low levels, as sweetness and/or
flavor enhancers. Examples include thaumatin and monk fruit extract. Woo explained trigeminal-on-taste “intramodal” sweetness enhancement using the examples of carbonation, a trigeminal pain agent, which can make artificial high-potency sweeteners taste more like sugar. It is labeled as “carbonated water.”

Beverages formulated with high-potency sweeteners have also shown in panels to taste sweeter at higher temperatures. Some studies have shown that the shape of a food,
specifically a more rounded shape—as in oranges or apples—tends to be associated with sweeter stimuli. For example, round chocolates were found to taste sweeter than other shapes.

Research has found that color influences sweetness, as well. Strawberry mousse was sweeter and more liked on a white plate than on a black plate. Hot chocolate tasted sweeter and had more aroma in a dark cream cup than in a white or red cup–“Why? I don’t
know,” smiled Woo.

Clean label, reduced-sugar foods and beverages with high-potency and bulk sweeteners can be made even sweeter with cross-modal correspondences. Woo concluded: “As Ernest Starling, 1866–1927, Nobel Prize winner and discoverer of the first hormone put it: ‘The physiology of today is the medicine of tomorrow.’”

Alex Woo, Ph.D., Managing Director and Founder, W2O Food Innovation,, +1.425.985.8168,


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