In the dietary supplement industry, laboratories must adhere to strict quality standards and regulations. As a requirement of the United States’ Code of Federal Regulation, all components of dietary supplements must meet a specified identity.¹ You must establish a specification for any point, step, or stage in the manufacturing process where control is necessary to ensure the quality of the dietary supplement and that the product is packaged and labeled as specified in the master manufacturing record. As such, the industry, in conjunction with the governing bodies, has had increased interest in identification methods. 
 
Identification Method for Milk & Whey Protein Concentrates
The United States Pharmacopeia Food Chemical Codex (USP-FCC) does not include monographs for many protein sources, including milk protein concentrate, while the monograph for whey protein concentrate does not list a single comprehensive identification method, but instead relies on a combined assessment of Ash (Total), Fat, Lactose, Loss on Drying, and Protein Content. An accurate protein identification assay is synonymous to prevention of economic adulteration in protein powders. An identification method is possible by studying the background bacterial DNA.²
 
Milk and whey proteins both contain significant background bacterial DNA.³ Due to the nature of dairy farming, background DNA of many bacterial species associated with the farming environment, skin, gut, and mammary glands remain even after these bacteria are destroyed by pasteurization.⁴ As a part of the cheese-making process, starter cultures are added to milk, and remain even after processing the whey byproduct.⁵ 
 
Traditional Sanger Sequencing for microbial identification sequences the 16S region of the ribosomal rRNA of bacteria, and researchers utilize a reference library to compare this sequence against known bacterial 16S rRNA sequences. The advent of next-generation sequencing (NGS) allows for significantly advanced DNA studies, but researchers can still utilize the 16S region and the curated libraries available. 16S Metagenomics uses NGS to sequence hundreds of thousands of individual 16S rRNA molecules in a given sample. Each molecule is then compared to a curated reference library, resulting in a complex microbiome profile of the sample in question. 
 
Each microbiome profile differs from other profiles in both alpha and beta diversity, specifically, how many and which species are present.⁶ Using these measurements, calculated by bioinformatics software, a principle coordinate plot can be created, where different groupings of samples are separated in space based on their differences in alpha and beta diversity. In this study, we use principle coordinate analysis to differentiate groupings of milk and whey protein concentrates, demonstrating microbiome profiling by 16S Metagenomics as an important forensic tool for differentiation of samples with background bacterial DNA content, and as a fully applicable identification method for milk and whey protein concentrates.
 
A Case Study: Testing a Method for Identification of Milk and Whey Proteins
Protein Powder Identification presents a challenge in Quality Control. There is currently deliberation of the specificity of methods for the identification of milk proteins, and the consensus identification method of whey protein from the USP-FCC relies on a combined analysis of the testing of Ash, Fat, Lactose, Loss on Drying, and Protein. 
 
Milk and whey proteins both contain significant background DNA content. Milk and whey proteins retain cow DNA, but also retain bacterial DNA. DNA from the natural flora of the cow, the dairy processing plant, and in the case of whey protein, the cheese-making procedure remain post-processing. By utilizing 16S Metagenomics, technology which enables researchers to study microbiomes, the retained bacterial DNA in protein powders can be sequenced and cross-referenced to a curated library and ultimately create a microbiome profile of these raw materials. This profile can be measured for both alpha and beta diversity, specifically how many and which species of bacteria are present. The microbiome profile of milk and whey proteins differ significantly. 
 
In this study “Microbiome Profiling of Milk and Whey Proteins by 16S Metagenomics,” Herbalife Nutrition researchers demonstrated that the microbiome profile of processed cow proteins can be used for raw material identification. Using 16S Metagenomics, we measure alpha and beta diversity of the microbiome profile of each protein powder and use principle coordinate analysis to produce differential groupings, providing a novel identification method for raw materials. This demonstrates microbiome profiling can be an important forensic tool for quality control.
 
Validation of Microbiome Profiling by 16S Metagenomics
This data was presented at the Association of Official Analytical Chemists (AOAC) International 2019 Annual Meeting and Exposition in Denver in September 2019, and validated that Microbiome Profiling by 16S Metagenomics can be an important forensic tool in Food and Dietary Supplement Quality Control. Each protein concentrate retains background DNA content reflective of their processing. Milk protein concentrate has biodiversity reflective of the dairy farm, processing plant, and cow flora. Whey protein concentrate has diminished biodiversity, due to the purposeful inoculation and overall abundance of cheese starter cultures of the family Streptococcaceae. These differences in Alpha and Beta Diversity can be seen using Principle Coordinate Analysis (PCoA) to differentiate protein sources. 

References

1. CFR – Code of Federal Regulations Title 21, 111.70, 2018 April 1. Part 111 — Current Good Manufacturing Practice In Manufacturing, Packaging, Labeling, Or Holding Operations For Dietary Supplements. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=111.70
 
2. C. M. Thompson, et al., “Development of a Differential Multiplex PCR Assay for the Supplemental Identification of Different Sources of Proteins.” AOAC International, 2019.
 
3. A. Pirondini et al., “Yield and Amplificability of Different DNA Extraction Procedures for Traceability in the Dairy Food Chain.” Food Control, 2010, 21: 663–668.
 
4. L. Quigley et al. “The Complex Microbiota of Raw Milk.” FEMS Microbiology Reviews, 2013, 37: 664- 698.
 
5. M. Morea et al., “Molecular and Physiological Characterization of Daminant Bacterial populationsinTraditionalMozzarellaCheeseProcessing.” AppliedMicrobiology,2001,87(4).
 
6. J. K. Goodrich et al., “Conducting a Microbiome Study,” Cell, 2014; 158(2): 250-262.