Protein Analysis: Methods, Reliability and Quality

The protein analysis is fundamental when it comes to assessing the quality of a product. This process should be done properly in order to be able to detect and prevent frauds

In HSN, we stand up for quality and transparency

That is why we also offer detailed analyses of our proteins in our website. Moreover, we have written this article in order to explain the methods that we have used as well as other techniques.

Importance of Protein Powder

At this point, everyone should know how important proteins are when it comes to nutrition. It is becoming clearer and clearer that taking a proper supply of protein in the diet is crucial for our health.

In the world of sports, proteins are even more important due to their role in supporting the muscle adaptations to exercise

Ideally, these proteins should come from food

Nevertheless, we cannot deny that protein powder is a highly nutritional product that makes it easier for us to take this nutrient. In fact, it becomes specially useful when we have lost our appetite or when we have problems to chew (like the elderly). Or perhaps it is just a matter of comfort, since it is very easy to carry and use.

The Importance of Protein Analysis

We have been able to witness how the commercialization of protein supplements has peaked in the last few years.

The existing competitiveness between manufacturers increases the number of products available with juicy discounts and adjusted prices

It is no surprise that this has also carried the rise of fraud which have seriously damaged the credibility of this sector. That is why, we feel like there is an obligation to show clear information from laboratory tests that show the methods that we have used in detail.

Why do a Protein Analysis?

Strategies to detect food fraud

There are two main ways of detecting potential frauds in the food industry¹:

Adulteration Analysis

In this case, a positive result would mean that there is a specific material missing. The problem of this method is that we have to know these substances previously.

Purity Analysis

This strategy is quite effective when the adulterant is high enough to detect a lack of purity from the original raw ingredient.

Foundations of Protein Analysis

Even though there are many techniques to analyze the protein content from food products, we will use this article to focus on explaining the most frequent analysis in the world of sport supplementation.

Protein information on the label

Usually, there are three types of information on the label of a protein product or supplement:

1. Source or sources of the protein: it comes in the ingredient list.
2. Amount of protein: see nutrient facts.
3. Amino acid profile of the protein: Aminogram.

Objective of the analyses

The objective of laboratory analyses will be to determine the amount of protein and amino acids from a specific source.

Determining the total protein content

The most common methods will determine the total nitrogen content. Then, it will extrapolate this information to the total protein content with several conversion factors.

To put it simply, these are indirect methods

Above all, this strategy relies on the fact that proteins are the only nutrients that provide nitrogen to the diet. However, the problem is that there are other non-protein compounds that also provide nitrogen.

Amino Spiking

This is the main reason why the methods to detect nitrogen are not reliable when it comes to fraud detection. In fact, one of the most common frauds is amino spiking, which will not appear on these analyses.

To put it simply, amino spiking consists of adding one or more cheap amino acids (amino acids contain nitrogen) to a low purity protein base.

Methods to detect total nitrogen

Kjeldahl

It is basically made up of four steps that digest the sample in order to measure the nitrogen supply (both from protein and non-protein sources).

To do so, this nitrogen becomes ammonia, which is isolated through a distillation process. Then, it determines the total nitrogen content indirectly through a series of chemical reactions.

Its main weakness lies in its inability to differentiate between protein a non-protein content.

To solve this problem, there is a previous process known as protein precipitation. This process will isolate the protein and measure the nitrogen that comes exclusively from this source.

Dumas or combustion method

This method relies in the formation of gaseous substances that result from the combustion of the sample. Consequently, it produces NOx and N2, among others. After reducing the NOx to N2, it determines the total N2.

Currently, it uses nitrogen analyzers that simplify, cheapen and make the procedure safer and faster than traditional methods such as Kjeldahl.

Conversion factors

Like we mentioned previously, we need to determine the amount of nitrogen before assessing the protein content.

Traditionally, people have relied on factor 6.25 in order to calculate the protein from nitrogen.

Above all, this is based upon the assumption that the nitrogen content from protein is a 16%

However, it is not that simple, since the nitrogen content changes depending on the type of protein. In fact, the amino acid profile will be different.

Despite their limitations, the most common factors are the ones proposed by Jones in 1931²

Limitations

Depending on the study and methodology, these factors can cause severe oscillations, even when we use the same type of protein.

Another big limitation is applying the factor 6.25 when we do not know the specific factor of a protein source. Consequently, this can result in over- and underestimations of the protein content.

The importance of the aminogram

Seeing that the methods to assess the total nitrogen are quite susceptible to manipulations, we will need more precise strategies that will help us unmask possible frauds.

What is the aminogram from a protein?

The aminogram is nothing else than the list of amino acids that make up a protein.

This allows us to carry two types of protein analysis:

1. Qualitative: due to the differentation of each one of the amino acids.
2. Quantitative: if we add on each amino acid we can obtain the total protein content.

Aminogram example

Here is a simple example for a whey protein and the amino acid glycine

According to the literature and the previous laboratory analyses, 100g of whey protein isolate should have between 1.6 – 1.8 grams of glycine.

If we analyze the aminogram and observe that 100g of product have much more glycine content than that, we will know that the protein has been adulterated. For instance, it could have been by adding more of this amino acid or blending the product with a cheaper protein.

How can we analyze the aminogram from a protein?

Without any doubt, one of the best techniques could be the HPLC (High Performance Liquid Chromatography).

With the HPLC method, we will obtain something that looks like this graphic:

Before the analysis, the sample is previously treated, so that it will be able to detect each amino acid individually

The basis of this technique is that each amino acid has different physicochemical properties. Changing the conditions of the analysis will allow us to detect these amino acids.

Interpretation of the analyses by HSNstore.com

If you have visited our website in order to purchase some of our products, you have probably seen this section:

External Laboratory

The reason why is that we have analyzed samples of many of our products and we wanted to share the results with you. The aim is no other than to show that we do not cheat, since transparency is one of our core values.

ENAC Accreditation

We have not done these analyses, in fact, they have been carried out by an independent laboratory. Moreover, this laboratory has the ENAC accreditation (Accreditation National Entity). Actually, you can find their seal in the documents from the analyses.

The ENAC literally defines the accreditation as: “an international tool used to generate confidence in the correct performance of a very specific type of activity called Conformity Assessment Activities. These include testing, calibration, inspection, certification and verification, among others.”

Example of an HSN Protein Analysis: 100% Whey Protein Isolate

Let’s see what information can we find in the analyses available in HSNstore.com. To do so, we will use the analysis of the whey protein isolate.

Protein content

First of all, we are going to see the total protein content according to the analyses.

If we start at the top, we will see the product description or reference (blue) with its corresponding lot number (green).

Method

The method used for this product has been the Dumas method (yellow) which we have explained previously. Therefore, they have determined the total nitrogen content to multiply it for the factors 6.25 or 6.38 and obtain the protein value.

Dry matter

Finally, we can see that the protein value has been calculated in dry matter after multiplying it for 6.38 (red).

Uncertainty

According to this analysis the protein value is of a 90.22%. Although we need to take into account that this value is an average. Therefore, the percentage may vary.

This variation is what is known as uncertainty

Comparison between the nutrient facts

This information corresponds to the amount of protein indicated in our label: 93g per 100g of product, a 93%.

What about the Aminogram?

But after reading the article, you may be thinking that the Dumas method is not useful when it comes to detecting frauds. In fact, that 935 could be the result of any type of adulteration.

Do not worry! That is why we have also provided the amino acid analysis from this protein:

Once again, we can find the reference and lot number of the product (blue and green respectively).

HPLC Analysis

If we focus on the analysis, the methodology corresponds to the ISO 13903:2005 international regulation or HPLC.

As we have previously seen, it is easy to detect frauds with this technique.

Then, if we combine all these numbers (which also have a level of uncertainty), the result is 94.99 grams of amino acids per 100g of product.

Therefore, the first test is satisfactory and it equals the values mentioned previously

This is an example of an aminogram from a study³:

Keeping in mind the inter-laboratory variations, we can see that the results are quite similar in general.

Comparing the aminogram and label

After all, we can see that the results are practically identical for all the amino acids

Evidently, there are small variations, since the analysis from our suppliers and the external laboratory may vary, Therefore, the label reflects the typical values of the product.

Conclusions

There are many other analyses available, all of them with their strengths and weaknesses. But we have chose the ones that seem to be more frequent. In HSNstore, we have a compromise with all our customers and we want to offer the best quality and transparency about our products.

Bibliography

1. Moore JC, DeVries JW, Lipp M, Griffiths JC, Abernethy DR. Total protein methods and their potential utility to reduce the risk of food protein adulteration. Compr Rev Food Sci Food Saf. 2010;9(4):330-357. doi:10.1111/j.1541-4337.2010.00114.x
2. Mariotti F, Tomé D, Mirand PP. Converting nitrogen into protein – Beyond 6.25 and Jones’ factors. Crit Rev Food Sci Nutr. 2008;48(2):177-184. doi:10.1080/10408390701279749
3. Sinha R, Radha C, Prakash J, Kaul P. Whey protein hydrolysate: Functional properties, nutritional quality and utilization in beverage formulation. Food Chem. 2007;101(4):1484- 1491. doi:10.1016/j.foodchem.2006.04.021
4. https://www.enac.es/

Related Entries

• Amino Spiking: What is it
• How to Compare Proteins (Whey Protein)