A review of current analytical methods for the determination of prebiotics in foods

Research Article
146 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
A review of current analytical methods for the determination 
 of prebiotics in foods
Tran Hung Son1,2*, Vu Thi Thanh An1, Mac Thi Thanh Hoa1
Cao Cong Khanh1, Le Thi Hong Hao1
1National Institute for Food Control, Hanoi, Vietnam
2Natural Product Informatics Research Center, Korea Institute of Science and Technology (KIST), 
Gangwon, Republic of Korea
(Received: 03/05/2021; Accepted: 24/06/2021)
Abstract
Prebiotics are a group that improves the human intestinal microbiota. The relationship 
between prebiotics and human health has been an area of increasing interest in recent years. 
Fructo-oligosaccharides and galacto-oligosaccharides are the two important groups of 
prebiotics with beneficial effects on human health. Besides that, more and more carbohydrates 
have shown a good potential to be prebiotics has been studied. This study presents a review of 
the characteristics, properties, and analytical methods used for the determination of prebiotics 
in food products. Most of the methods found using chromatographic methods, especially 
HPAEC - PAD but also another method, such as spectrometric methods have been presented. 
A discussion was presented highlighting the drawbacks of current analytical methods and the 
need to develop these methods to analyze the complex food samples containing prebiotics. 
Keywords: prebiotics, review, foods, analytical methods, fructo-oligosaccharides, galacto-
oligosaccharides.
1. INTRODUCTION 
Prebiotics are known as non-digestible carbohydrates selectively fermented to stimulate 
microbiota in the human intestinal. In 1995, prebiotics was firstly defined by Gibson and 
Roberfroid as “a non-digestible food ingredient that beneficially affects the host by selectively 
stimulating the growth and/or activity of one or a limited number of bacteria in the colon, 
and thus improves host health” [1]. In 2008, the 6th Meeting of the International Scientific 
Association of Probiotics and Prebiotics (ISAPP) defined “dietary prebiotics” as “a selectively 
fermented ingredient that results in specific changes in the composition and/or activity of the 
gastrointestinal microbiota, thus conferring benefit(s) upon host health” [2].
There are some criteria used to classify a compound as a prebiotic. Firstly, it should be 
resistant to acidic pH of stomach, cannot be hydrolysed by mammalian enzymes, and also 
should not be absorbed in the gastrointestinal tract. Secondly, it can be fermented by intestinal 
microbiota, and finally, the growth and/or activity of the intestinal bacteria can be selectively 
stimulated by this compound and this process improves the host’s health [2]. Some prebiotics 
can be extracted from plant sources, but most are synthesized commercially using enzymatic or 
chemical methods. Overall, prebiotics are manufactured by four major routes: direct extraction, 
*Corresponding author: Tel 0389811395 Email: tranhungson113@gmail.com
147Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Tran Hung Son, Vu Thi Thanh An, Mac Thi Thanh Hoa,... Le Thi Hong Hao
controlled hydrolysis, trans-glycosylation, and chemical process [3]. 
Most of the prebiotics are carbohydrates and two criteria can be applied to distinguish 
fiber from carbohydrate-derived prebiotics. Firstly, fibers are carbohydrates with a degree 
of polymerization (DP) equal or higher than three. Secondly, endogenous enzymes in the 
small intestine cannot hydrolyse them. It should be taken into account that fiber solubility or 
fermentability is not crucial. The most common prebiotics are inulin, fructo-oligosaccharides/
oligofructose (FOS), β-glucan, galacto-oligosaccharides (GOS), however, other complex 
saccharides, and fibers have been recently considered as prebiotics [4]. 
Prebiotics has a beneficial effect on human health, and has greater potential for supplement 
into a broader range of common food products as they have both technical and nutritional 
properties. They act as food sources for the growth of probiotic bacteria (bifidobacteria and lactic 
acid bacteria), thus enhance the gastrointestinal functions and immune system. Prebiotics also 
increase the absorption of calcium and magnesium, reduce blood glucose, and lipid levels. [5]. 
Nowadays, food products that contain prebiotics are becoming more and more popular and 
diverse. Therefore, the application of methods to analyse the content of prebiotics in the above 
products to evaluate product quality is very important and necessary. Due to the importance 
of prebiotics in food products, this paper presents a review of the classification of prebiotics, 
their properties and the most important - the analytical methods and techniques used for the 
determination of prebiotics in foods. Among them, spectrophotometric and chromatographic 
methods are most commonly used in the quantification of prebiotics.
2. TYPES  ... he 
degree of polymerization of prebiotic materials. The fact that different prebiotics can provide 
fragments with the same m/z value will lead to difficulty in characterizing the prebiotics present 
in the matrix. Therefore, the application of the above methods for the direct quantification of 
prebiotics in products requires further investigation and improvement in sample preparations.
4.2. The disadvantages of current analytical methods
In general, the methods of analysis of prebiotics are currently studied based on different 
analytical techniques and many methods have been approved by AOAC and AACC. However, 
the application of these methods in the analysis of food products containing faces many 
difficulties. The specificity of the method in the analysis of products containing mixed prebiotics 
is a problem. With the general principle of using enzymes to hydrolyze prebiotics in polymer 
form to monomers and then, quantification is based on the determination of monomers content. 
The presence of free monomers (usually monosaccharides) in the matrix leads to positive errors, 
thus requiring simultaneous analysis of both hydrolyzed and non-hydrolyzed samples, resulting 
in complex and time-consuming analytical procedures.
On the other hand, the fact that an enzyme can have hydrolytic activity against two prebiotics 
with similar monomers makes it extremely difficult to identify each prebiotics individually, such 
as a sample containing both Inulin and FOS. In addition, the complexity of the samples also 
troubles the analysis. For example, in dairy products containing small amounts of TGOS, a large 
amount of lactose (up to 50%) in milk is hydrolyzed simultaneously with TGOS by the enzyme 
lactase, leading to very large errors in the calculation of results. The problem of LOD and LOQ 
of the method also needs to be discussed. Currently, analytical methods with high quantitative 
limits are difficult to meet in analyzing products containing only a small amount of prebiotics. 
The presence of many other compounds also affects the analytical process.
156 Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
A review of current analytical methods for the determination of prebiotics in foods
5. CONCLUSIONS 
Prebiotics, which have shown many positive effects on human health, especially the 
improvement in human intestinal microbiota. This review paper has shown that a variety of 
analytical methods for the identification and determination of prebiotics have been developed 
and described. The most common analytical methods and techniques used for determination of 
prebiotics group are spectrometric methods and chromatographic methods. Spectrophotometric 
methods have several advantages, such as good analytical specificity and they are easier, less 
expensive compared with the other methods. However, the spectrophotometric methods are 
less sensitive than other methods. Chromatographic methods, especially HPAEC-PAD are most 
commonly used for the quantification of prebiotics. Chromatographic methods generally provide 
a lower limit of quantification. However, the key issue in the use of chromatographic methods 
is the separation of the monosaccharides in the mixture after hydrolysis of prebiotics. A major 
problem with current analytical methods is their efficiency in analyzing a mixture of prebiotics 
present in food samples. In addition, the removal of other compounds which hydrolyzed along 
with prebiotics in the sample preparation step is also an unresolved problem. Therefore, in the 
following years, it is very important to develop quantitative analytical methods for prebiotics 
in food that can provide a useful tool for assessing the quality of food products that contains 
prebiotics.
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159Vietnamese Journal of Food Control, Vol. 4, No. 2, 2021
Tran Hung Son, Vu Thi Thanh An, Mac Thi Thanh Hoa,... Le Thi Hong Hao
Tổng quan các phương pháp xác định hàm lượng 
 prebiotic trong thực phẩm
Trần Hùng Sơn1,2*, Vũ Thị Thanh An1, Mạc Thị Thanh Hoa1 
Cao Công Khánh1, Lê Thị Hồng Hảo1
1Viện Kiểm nghiệm an toàn vệ sinh thực phẩm quốc gia, Hà Nội, Việt Nam
2Trung tâm Nghiên cứu Thông tin Sản phẩm tự nhiên, Viện Khoa học và Công nghệ Hàn Quốc, 
Gangwon, Republic of Korea
Tóm tắt
Prebiotic là một nhóm các chất có khả năng cải thiện hệ thống vi sinh vật đường ruột của 
con người. Sự tác động của các prebiotic đối với sức khỏe của con người là một lĩnh vực ngày 
càng được quan tâm và nghiên cứu sâu trong những năm gần đây. Fructo-oligosaccharides và 
galacto-oligosaccharides là hai loại prebiotic chính được chứng minh có nhiều tác động có lợi 
đối với sức khỏe của con người. Bên cạnh đó, ngày càng có nhiều hơn các phân tử carbohydrate 
cho thấy tiềm năng của một prebiotic. Bài báo này tổng quan sự phân loại, đặc điểm và các 
phương pháp phân tích prebiotic có trong thực phẩm. Phần lớn các phương pháp hiện nay được 
sử dụng là phương pháp sắc ký, đặc biệt là HPAEC-PAD và đồng thời còn có các phương pháp 
hữu ích khác như phương pháp quang phổ. Bài báo cũng chỉ ra những vấn đề đang tồn tại trong 
các phương pháp phân tích hiện nay và nhấn mạnh sự cần thiết trong việc phát triển các phương 
pháp phân tích để định lượng prebiotic trong các nền mẫu thực phẩm phức tạp. 
Từ khóa: prebiotics, tổng quan, thực phẩm, phương pháp phân tích, fructo-oligosaccharides, 
galacto-oligosaccharides.