Chapter 1: Overview of Protein Flavours

The United Nations has predicted that the global population will reach 9.7 billion in 2050 and 10.4 billion in 2100.¹  Protein is a critical nutrient and an important food ingredient. Due to population growth, there is an increasing demand for high-quality proteins. Moreover, the increase in consumers’ awareness of health and fitness, alarming rise in obesity rates, increase in disposable income, and increased demand from the millennial population for supplements are driving the growth of the protein ingredient market.

With the increasing demand and popularity of replacing meat and seafood with alternative proteins, high-quality protein ingredients with good manufacturing functionalities and flavours are highly sought after. A good number of studies have been done to investigate the functionalities of protein ingredients (e.g., solubility, foaming, emulsifying, gelation, water holding, and oil absorption). However, the flavour and protein–flavour interaction of protein ingredients under various processing conditions and in different formulations have not been well investigated. It is known that the characteristic aroma of plant proteins and the allergenicity of some plant proteins restrict their wide applications. For example, the green and beany odours from soy and pea proteins are described as off-flavour by consumers. Profiling the flavour compounds and removing or masking the odorants responsible for the off-flavours would be critical in product development and product quality control. This book aims to consolidate much-needed information on the flavour of food protein ingredients.

The global protein market has grown strongly in the past decade and is projected to reach over US$114 billion by 2030 (Table 1.1).²  Among the protein types, the demand for plant proteins has been steadily increasing due to religious reasons, rising health awareness among consumers, and environmental concerns. The global plant protein market value was estimated to be US$12.2 billion in 2022.^3,4  It has been forecast to achieve US$17.4 billion by 2027 by Markets and Markets,⁴  US$17.8 billion by 2027 by Statista,³  and US$22.7 billion by 2031 by Transparency Market Research.⁵  Especially, plant-based protein substitutes are a fast-growing section.⁶  Plant-based meat had a market value of US$6.67 billion in 2020 and is expected to reach US$16.7 billion in 2026.⁶  Globally, the number of companies producing plant-based dairy, meat, and egg alternatives has increased from less than 40 in 2010 to more than 160 by 2022.¹⁶  In the United States, sales of plant-based alternatives amounted to US$5.8 billion, with about 43% (US$2.5 billion) coming from milk alternatives, 24% (US$1.4 billion) from meat alternatives, and 0.46% (US$27 million) from egg alternatives.¹⁶ 

Protein supplements are a major form of proteinaceous products. The global protein supplements market was valued at US$6.26 billion in 2021 and is expected to grow at a compound annual growth rate (CAGR) of 8.0% from 2021 to 2028.⁷  In the United States, the market for protein supplements was US$1.9 billion in 2019. It is expected to reach US$3.6 billion by 2028, at a CAGR of 7.7%.¹⁷  Protein supplements are available in the form of powder, ready-to-drink protein shakes, and protein bars. The most common protein supplements on the market are whey, soy, casein, and egg protein powders. Besides being used as protein supplements, proteins have been used as an ingredient in various products to enhance the protein content and as fat or egg replacers.^18–23 

The dairy protein market comprises milk protein, milk powder, whey protein, and caseinate. Whey protein, a by-product of cheese production, is the main dairy protein product available on the market. It has been used as a protein supplement in the form of protein isolate and concentrate. Whey protein also pioneered the market for protein bars, protein shakes, etc. According to Vantage Market Research, the whey protein market was estimated to be worth approximately US$9.7 billion in 2021 and is expected to reach $16.4 billion by 2028. The continued growth is driven by the demand for whey protein in nutritional supplements, personal care, and the food and beverage industry.⁸ 

Among plant proteins, soy protein has the largest market share at 64.4% and has gained a steady increase in the past decade.²⁴  The global soy protein market reached nearly US$9.19 billion in 2021 and is estimated to reach US$15.65 billion by 2027.⁹  The main demand for soy protein has been observed for bakery and functional foods. Other uses include pharmaceuticals, meat additives, and feed, among others. Other major plant proteins include wheat and pea proteins. The pea protein market size was estimated at US$213.1 million in 2020 and is projected to grow at a CAGR of 12.7% between 2021 and 2028.¹⁰  The major applications of pea protein are in dietary supplements, bakery products, meat substitutes, and beverages. Besides, protein powders of rice, almond, fava bean, broad bean, mung bean, hemp, etc. have been available in the market. Rice protein is predicted to reach a global market size of US$218.48 million by 2028.¹¹  The drive for using rice protein is the rising need for dietary foods that are gluten-free, non-genetically modified organisms (GMOs), and hypoallergenic products. The mung bean protein market was estimated to be US$226.6 million in 2022.¹²  Fava bean protein had a global market size of US$56.7 million in 2022.¹³  The Good Food Institute has curated an extensive list that features more than 1400 companies specializing in alternative protein production.²⁵  These companies cover a range of products, including plant-based, cultivated, fermented, and molecular-farmed options. As shown in Figure 1.1, the majority of these innovative companies focus on plant-based protein alternatives.

Novel proteins, such as edible insect proteins and algal proteins, are on the rise due to their sustainable production and environmental benefits. According to Research and Markets, the edible insect market is expected to reach US$9.60 billion by 2030, at a CAGR of 28.3% during the forecast period of 2022–2030.¹⁴  The volume of the market is predicted to reach 3.14 million tons by 2030. The algae protein market size exceeded US$700 million in 2021 and is projected to grow at over 6% CAGR between 2022 and 2028.¹⁵  The market growth is driven by the increasing product adoption in dietary supplements, food, and beverages.

Flavour is a synthesis of three chemical senses: taste, smell, and chemesthesis. It is one of the most important sensory properties of foods, along with colour and texture. Protein ingredients play an important role in food texture, which is related to their gelling, foaming, emulsifying, water-holding, oil-absorption, and dough-formation properties. Protein ingredients could affect the colour of food products through their natural colours. For example, whey and most plant proteins are white to slightly yellow in colour while, hemp protein is dark brown. Also, proteins could undergo a Maillard reaction during the processing and/or storage of the products to form dark pigment melanoidins.²⁶ 

Despite protein’s effect on other sensory properties, the undesirable flavour of protein ingredients has been a significant concern for product quality and a limiting factor for the use of protein ingredients in foods. For example, the ‘gluey’ flavour of stored casein was studied in the 1960s.²⁷  The removal of beany flavour from soy and pulse proteins has been studied for decades and is still an active research topic today.^28–30  The following discussion will focus on the flavour, especially the taste and odour, of protein ingredients.

Sweet, salty, sour, bitter, and umami are the five basic tastes. In general, the tastes of food proteins are contributed by peptides (bitter, sweet, and umami) and amino acids (bitter, umami, sweet, salty, and sour) derived from protein hydrolysis.³¹  Among the amino acids, l-glutamate and l-aspartate are known for having an umami sensation. In general, l-glycine, l-alanine, l-glutamine, l-proline, l-serine, and l-threonine have a sweet taste, while l-histidine has a bitter taste.³² 

Bitterness is a noticeable problem in protein hydrolysates or peptides. Enzymatic hydrolysis is a popular method of generating bioactive peptides and protein hydrolysates with improved functionalities.^33–35  Besides, hydrolysis can effectively lower the allergenicity of many food allergens, making it a very useful technique to produce hypoallergenic products and infant foods.^36–38  However, bitterness in the peptides is a hurdle to consumer acceptance. The taste of hydrolysates varies depending on the protein source, hydrolysis conditions, and proteolytic enzyme used.³⁹  It has been reported that several factors could determine the bitterness of peptides (e.g., peptide chain length, hydrophobicity, and Q-parameter). The intermediate peptides with no more than 8 amino acid residuals have shown the strongest bitterness. Neither amino acids nor long-chain peptides are the major causes of bitterness. More importantly, the hydrophobicity of the peptides has a strong correlation with their bitterness. Another factor is the Q-parameter. It is defined as the average amount of free energy needed to transfer amino acid chains from ethanol to water and expressed in cal mol⁻¹. The Q is correlated to the hydrophobicity of the composing amino acids.³⁹  In an effort to reduce the bitterness in protein hydrolysates, 24 bitter taste inhibitors were documented for whey protein hydrolysate.⁴⁰  Many of the bitter taste inhibitors are sugars, salts, or nucleotides.

Besides the bitter taste from peptides and amino acids, some phytochemicals (e.g., saponin, phenolic compounds, and alkaloids) and bitter lipids remaining in protein products could also be the source of the bitter taste (e.g., many studies have reported saponin as a source of bitterness in pea protein).^41–43  Also, bitter-tasting lipids were identified from commercial pea protein isolates in a recent study using ultra-high-performance liquid chromatography–differential ion mobility spectrometry (DMS)–tandem mass spectrometry (MS).⁴⁴ 

Isolated protein ingredients from meat, dairy, eggs, and plant sources are generally considered bland in taste. A salty and sour taste could be noticeable in proteins due to the charged amino acid side chains.⁴⁵  But in general, salty and sour tastes are not significant in proteins or peptides. Some proteins derived from tropical plants have an intensely sweet taste (e.g., thaumatin,⁴⁶  monellin,⁴⁷  and brazzein⁴⁸ ). Besides, miraculin is a taste-modifying protein that makes acids taste sweet when the taste buds are exposed to it.⁴⁹  However, miraculin itself is sensory inactive. It activates the sweet taste receptor only at acidic pHs when a sour solution or compound co-exists.

In protein isolates, mild yet significant odours exist due to the oxidation of residual lipids and amino acids, degradation of amino acids, and Maillard reaction of proteins with reducing sugars. Compared with tastes, odour sensations are much more complicated and difficult to describe. The odour of whey protein was described as sweet (desirable), cardboard (undesirable), brothy, cucumber, etc.^50–55  Stored casein was found to have a gluey off-odour.⁵⁶  Soy and pulse proteins have a strong beany odour.^{28,30,57–59}  Soy odours were described as beany, corn meal, musty, and toasted.⁶⁰  Most vegetable proteins also have a green or grassy off-odour.⁶¹  Su et al. reviewed the odour lexicons used for dairy ingredients and the volatiles associated with the odours.⁶²  The sensory lexicon of whey and soy proteins was developed by Russell et al., and that of pulse proteins was reviewed by Chigwedere et al.⁶³ 

The describers people use to describe odours typically correspond to a mixture of compounds. For example, the cardboard flavour that was reported in whey protein is a combination of pentanal, heptanal, nonanal, 1-octen-3-one, and dimethyl trisulphide.⁶⁴  Even though compounds of similar chemical structures tend to have similar odours, chemicals from the same group could give off different odours.⁶²  In general, the major groups of volatile compounds found in protein isolates include aldehydes (e.g., hexanal that has a green and grassy aroma), ketones (e.g., 2-nonanone with a fresh, green, weedy, and earthy smell), alcohols (e.g., hexanol with a herbaceous, woody, and green aroma), and sulphur-containing compounds (e.g., dimethyl disulphide with a garlic-like odour).^65–67  A comprehensive description of the odour characteristics of various flavour ingredients can be found in Fenaroli’s Handbook of Flavor Ingredients.⁶⁸  In this section, we only aim to provide a description of the odour characteristics of various food proteins. The volatile compounds associated with odours and off-odours will be discussed in later chapters.

The odour of protein ingredients is complicated by the numerous factors that could influence odour formation in protein ingredients. The major factors include the protein source (inherent odours), preparation (extraction and drying) methods, and storage conditions. Protein isolates derived from different foods (e.g., whey, egg, soy, pea, and mung bean) have a volatile profile that is unique to the food of origin. Soy and pea proteins have a noticeable beany flavour, while rice proteins have a high content of sulphur-containing volatiles.^57,65  Besides, proteins derived from the same food could have varied flavour properties due to the different starting materials. Smith et al. examined the effects of the source of whey (whey derived from cheddar, mozzarella, cottage cheese, and rennet casein) on the volatile profiles of whey protein isolates.⁶⁹  Their study demonstrated the changes in volatile compounds and their abundance due to the different sources of whey.

Preparation methods play a significant role in flavour formation. They often determine the physical (e.g., particle size and thermal treatment history) and chemical (e.g., residual lipid content and amount and activity of lipoxygenase) properties of the end protein product, therefore influencing its flavour profile. For example, several studies examined the drying methods and conditions on the flavour of derived protein products.^70–74  In whey protein concentrates, a low feed concentration of 10% led to increased overall aroma, intensified cabbage and cardboard off-odours, and higher concentrations of certain aldehydes (pentanal, hexanal, heptanal, decanal, (E)2-decenal, and 2,4-decadienal) and sulphur-containing volatiles (dimethyl trisulphide and dimethyl disulphide) as compared to products spray dried at 25% feed solids. The drying temperature is another key parameter. Product spray dried at a lower inlet temperature showed increased cardboard flavour compared to product spray dried at a higher inlet temperature.⁷⁰  Different processing techniques are also utilized to inhibit the formation of off-odours in protein isolates. These methods can be grouped as physical, chemical, and biological techniques. Physical methods include grinding, heating, and exposure to microwaves, magnetic fields, electric fields, irradiation, etc. Novel techniques in this area are constantly emerging, which provide new tools for protein and flavour modifications. The most commonly used chemical modifications utilize pH adjustment, organic solvents, reductants, supercritical carbon dioxide extraction,⁷⁵  etc. Biological methods include a wide range of approaches such as fermentation, germination, enzymatic treatment, and genetic engineering. The applications of some of these techniques to improve the flavour of pulse and soy protein isolates have been reviewed.^30,76,77 

The storage conditions, e.g., temperature and water activity, also affect the flavour formation and stability of protein ingredients. Storage under elevated temperatures and high water activity conditions has been reported to increase the concentrations of volatile compounds in whey⁷⁸  and pea proteins.⁷⁹  For example, Mehle et al. reported that pea protein isolates stored at 37 °C and 0.501 water activity conditions had a greater degree of aroma change compared to samples stored at refrigerated temperatures and lower water activity conditions. The aroma changes were related to the generation of 1-octen-3-ol and 2-4-decadienal, which were products of lipid oxidation.⁷⁹ 

As protein ingredients are widely used in various food products, especially plant-based meat alternatives, a better understanding of the flavour of the protein ingredients and their impact on the final products is much needed. The flavour of various food proteins and how they are affected by processing and storage conditions are covered in detail in Chapters 5 to 10.

Besides the inherent and environmental factors that influence flavour formation in food proteins, analytical and sample preparation methods could also affect the odours detected. The commonly used tools to study food aroma include sensory evaluation, gas chromatography (GC)-MS, GC-olfactometry (O), GC-O-MS, GC-flame-ionization detection (FID), electronic nose, etc. In GC-O-MS, the volatile compounds coming out of the GC column are simultaneously detected by MS and human participants at the olfactory detection port (ODP). This technique enables the identification of aroma-active compounds and the association of aroma characteristics with volatile compounds. The applications of GC-O-MS in flavour analysis were reviewed by Song and Liu.⁸⁰  In recent years, GC-ion mobility spectrometry (IMS) has been used in food flavour analysis to detect food adulteration, spoilage, and off-flavours.⁸¹ 

The most direct way to get samples for volatile analysis is equilibrium/static headspace (HS).^65,82,83  This method enables the detection of an unaltered volatile profile from a sample headspace. However, its sensitivity is very low.⁸⁴  Methods to concentrate HS volatiles include dynamic HS (DHS, also known as purge and trap),^85,86  solid-phase microextraction (SPME),^87–89  in-tube extraction (ITEX), sorptive extraction,⁹⁰  etc. The analytical methods for detecting volatile compounds and flavour–protein interactions are discussed in detail in Chapter 2. In general, the sampling method and conditions, e.g., extraction time, extraction temperature, extraction medium (pH and ionic strength), fibre, agitation, preheating time, adsorption time, adsorption temperature, desorption time, desorption temperature, injection depth, water content, and sample weight in the SPME method, could influence the analytical results. Singh et al. reported that increasing pH from 3 to 11 decreased recovery of volatiles while adding 1% NaCl increased the recovery.⁶¹  Therefore, it is critical to understand the differences in the sampling techniques to properly choose the method for the targeted samples and interpret the results.

Raw meat is often described as having an unpalatable, smelly, and salty flavour.⁹¹  However, when meat is cooked, it develops a pleasant aroma due to various thermal reactions. Non-volatile compounds, such as free amino acids and peptides, play a vital role in creating the desirable volatile compounds responsible for the meaty aroma.^92,93  Understanding the mechanism of meat flavour formation can aid in the development of meat-like aromas using non-animal proteins. Proteins, peptides, amino acids, lipids, sugars, and thiamine are all important components involved in the formation of meat flavours. Myofibrillar and sarcoplasmic proteins can bind to these flavour precursors and influence the release of flavour compounds.⁹⁴  Postmortem ageing has a positive impact on meat flavour precursors by increasing the breakdown of lipids and the release of small peptides.⁹⁵ 

When meat is heated, these precursors undergo a complex array of chemical reactions. These reactions include lipid oxidation, sugar degradation, thiamine degradation, thermal degradation of amino acids and peptides, the Maillard reaction, and interactions between lipid oxidation and the Maillard reaction.⁹³  The proteinaceous precursors are specifically involved in the latter three reactions. When amino acids and peptides are heated above 125 °C, they can undergo decarboxylation and deamination reactions, resulting in the degradation of these compounds into amines, aldehydes, hydrogen sulphide, and aromatic compounds. Cysteine and cystine have been identified as key contributors to the formation of meat-like volatile compounds, particularly thiazoles.^96,97  Pyrazines, which significantly contribute to the meat flavour, can be produced from serine and threonine degradation.^98,99  During the Maillard reaction, the free amino groups of amino acids, amines, peptides, and proteins react with reducing sugars, leading to the formation of various Maillard reaction products. These products include alcohols, aldehydes, furans, ketones, pyrazines, pyridines, pyrroles, sulphides, and thiophenes, which collectively contribute to the meat flavour.¹⁰⁰  The interaction between lipid oxidation and Maillard reaction compounds can give rise to additional volatile compounds, such as oxazoles, pyrazines, pyridines, thiazoles, and thiophenes with alkyl side chains. These compounds contribute to the more pronounced aroma of cooked meat.¹⁰¹ 

Peptides obtained through enzymatic protein hydrolysis can be utilized to generate Maillard reaction products with unique flavour profiles. Peptides possess a wide range of taste characteristics, such as sweet, bitter, umami, sour, and salty tastes.⁴⁵  Furthermore, peptides can contribute to a taste-enhancing phenomenon known as kokumi, which is often described as imparting thickness, mouthfulness, and richness to the taste experience.^102,103  A variety of animal and plant proteins, as well as proteases, can be utilized to produce these peptides.¹⁰⁴  The use of exopeptidases has demonstrated a successful reduction of bitterness in peptides derived from animal and plant proteins.¹⁰⁵ 

Maillard reaction products can be generated through the reaction between peptides and reducing sugars in various systems, including dry, wet, and organic solvent systems. Among these, the wet system is the most commonly employed method due to its ease of operation, low cost, and sustainability.⁹⁴  The flavour outcome of the Maillard reaction products can be influenced by factors such as peptide length, amino acid composition and sequence, and the susceptibility of peptide bonds to hydrolysis.¹⁰⁶  Furthermore, the flavour profiles can be manipulated by adjusting variables such as the type of reducing sugars and other additives, pH levels, temperatures, and heating durations. These factors allow for the creation of diverse flavour profiles in the Maillard reaction products.⁹⁴ 

The Maillard reaction products derived from peptides can exhibit a flavour-enhancing effect. For instance, the reaction between xylose and soybean peptides within the molecular weight range of 1–5 kDa has been found to enhance umami taste and kokumi sensation, indicating the significant potential of Maillard reaction products as taste enhancers.^107,108  Peptides within this specific molecular weight range have been shown to produce Maillard reaction products with reduced bitterness.^102,109  Similarly, the Maillard reaction between chicken peptides and xylose at 80 °C and with an extended heating duration resulted in the formation of products with low bitterness and a rich broth-like taste.¹⁰²  During the Maillard reaction between soybean peptides and xylose, peptide degradation and cross-linking occur simultaneously, with the cross-linking products contributing to the kokumi effect.¹⁰⁹  Enzymatic cross-linking of soybean peptides using transglutaminase can also generate Maillard reaction products that enhance mouthfulness and reduce bitterness.¹¹⁰  Furthermore, Maillard reaction products prepared from soybean peptides and various carbonyl compounds have been found to influence human perception of salty taste.¹¹¹  Additionally, taste-active cyclic dipeptides, known as 2,5-diketopiperazines, generated from the Maillard reaction have been identified to elicit astringent, salty, metallic, or bitter tastes, and have been detected in different processed foods.¹¹² 

The Maillard reaction products of peptides possess a strong aroma that is commonly associated with meat. In comparison with amino acids, peptides exhibit a greater ability to generate specific volatile compounds, including pyrazines, pyrazinones, thiazoles, and thiophenes.¹¹³  Peptides participate in various pathways of the Maillard reaction, such as bond cleavage, cyclization, and glycation.^102,114  The formation of specific volatile compounds during the Maillard reaction heavily relies on the molecular weight distribution and the composition and configuration of peptides.¹¹⁰  For instance, pyrazines responsible for the nutty and roasted meat aroma are more effectively produced by low molecular weight peptides (<500 Da) due to the high reactivity of amides in the reaction.¹⁰²  The structural characteristics of the N-terminal amino acid in peptides also influence the production of pyrazines.¹¹⁵  Additionally, pyrazinones, which possess a distinct toasted aroma, are unique Maillard reaction products derived from the reaction between dicarbonyls and glycine dipeptide. They cannot be formed in a system containing free amino acids alone.^116,117  Apart from pyrazines and pyrazinones, sulphur-containing heterocyclic compounds, such as thiophenes and thiazoles, formed during the Maillard reaction or Strecker degradation, play a crucial role in producing the roasted and meaty aroma.¹¹⁸  The release of hydrogen sulphide from sulphur-containing amino acids during the Maillard reaction leads to the formation of various sulphur-containing compounds with low odour thresholds when they react with Maillard reaction products.¹¹³  However, peptide-bound cysteines generate fewer thiazoles compared to free cysteines during the Maillard reaction. This is attributed to the inability of peptides to undergo typical Strecker degradation due to the absence of a free carboxyl group at the α-carbon in relation to the free amino group, which hinders decarboxylation.^100,113,119 

The Maillard reaction offers a valuable approach for the industrial production of meat flavourings, which are considered natural and green alternatives to traditional synthetic meat flavourings.^120,121  Protein hydrolysates contain a large number of peptides, which can result in a wide range of flavours that closely resemble the flavours of beef, chicken, pork, mutton, shellfish, salmon, cod, squid, and cocoa. They can also function as flavour-enhancing condiments.⁹⁴  These flavourings find applications in both meat products and plant-based meat analogues.

In meat products, the selection of flavourings depends on whether they are intended for low-temperature or sterilized meat products. For low-temperature meat products, it is crucial to choose flavourings with strong aromas and high volatility at low temperatures.^122,123  On the other hand, sterilized meat products undergo high-temperature treatment, which can lead to the loss of original flavours and the development of off-flavours. Therefore, it is essential to select flavourings with good heat stability or those generated through high-temperature reactions to enhance the flavour of sterilized meat products.^122,123 

When it comes to plant-based meat analogues, flavours can be incorporated by either adding flavour precursors¹²⁴  or directly incorporating the generated meat flavourings.¹²⁵  This enables the plant-based products to acquire meat-like flavours, thus enhancing their appeal to consumers.

Over the past decades, protein markets have been rapidly expanding due to the increased world population and consumer needs for nutritious foods. Proteins are important for the flavour of food products due to their unique inherent taste and odour, their capability to bind with other flavouring compounds, and their role as flavour precursors. Characterizing and improving the flavour characteristics of protein ingredients and understanding the effects of protein ingredients on the flavour of the final products are critical for product formulation and the creation of high-quality food products.

CAGR

Compound annual growth rate

DHS

Dynamic headspace

DMS

Differential ion mobility spectrometry

FID

Flame-ionization detection

GC

Gas chromatography

GMO

Genetically modified organism

HS

Headspace

IMS

Ion mobility spectrometry

ITEX

In-tube extraction

MS

Mass spectrometry

O

Olfactometry

ODP

Olfactory detection port

SPME

Solid-phase microextraction