Food digestion does not begin in the stomach. From the moment food enters the mouth, chewing, saliva and contact time begin to modify the food matrix and the compounds contained within it.
A recent study published in LWT – Food Science and Technology investigated this process in broccoli, exploring how human saliva and mechanical processing during the oral phase can influence the release and transformation of bioactive compounds.
The study focuses particularly on glucosinolates, characteristic compounds found in vegetables from the Brassicaceae family, and on the products formed from their transformation, including isothiocyanates (ITCs).
What happens to glucosinolates when we chew?
Glucosinolates occur naturally in broccoli, but their transformation begins when the structure of plant cells is disrupted.
During chewing, mechanical disruption allows glucosinolates to come into contact with an enzyme naturally present in plant tissue: myrosinase.
This interaction can give rise to several degradation products.
Among them are isothiocyanates, bioactive compounds that have been widely studied for their potential beneficial health properties.
However, the reaction does not always lead to the same products. Depending on the conditions present in the mouth, nitriles, thiocyanates and sulfur-containing compounds can also be formed.
It is precisely this balance between different transformation pathways that the study aimed to understand.
How was oral digestion simulated?
The researchers developed an ex vivo experimental model using human saliva.
Freeze-dried raw broccoli florets were used and subsequently rehydrated, helping to preserve the structure of the plant tissue and reduce transformations before the experiment began.
The team tested different proportions of saliva and water:
- 0% saliva;
- 50% saliva;
- 75% saliva;
- 100% saliva.
Different contact times of 1, 3 and 5 minutes were also tested, together with different mechanical processing times similar to chewing of 0, 1, 3 and 5 minutes.
In total, 24 different experimental conditions were analysed.
Figure 1. Representation of the experimental model and the main glucosinolate transformation pathways during oral digestion. Source: Gudino et al. (2026), LWT – Food Science and Technology.
Saliva quantity influences isothiocyanate formation
One of the most relevant findings of the study was that saliva proportion had a significant impact on total isothiocyanate formation.
When only water was used, the average total amount of ITCs was relatively low.
With a 50% saliva mixture, average ITC formation increased markedly, reaching the highest mean value among the conditions analysed.
The 75% saliva condition also showed high levels.
Interestingly, when 100% saliva was used, the amount of detected ITCs decreased again.
This shows that the relationship between saliva and food is more complex than a simple matter of hydration.
More saliva does not necessarily mean greater formation of bioactive compounds.
The authors suggest that components present in saliva, including proteins, may interact with isothiocyanates.
Because ITCs are highly reactive molecules, these interactions may reduce the amount that remains free and available for experimental detection.
At the same time, saliva composition may also favour other biochemical pathways involved in glucosinolate transformation.
Longer chewing also changes the outcome
Chewing plays a fundamental role because it increases the disruption of plant cells.
The greater the tissue disruption, the greater the contact between glucosinolates and the enzymes responsible for their transformation.
However, the study showed that extending processing time does not necessarily mean producing more isothiocyanates.
However, the study showed that extending processing time does not necessarily mean producing more isothiocyanates.
With longer contact and processing times, their amount may decrease due to their reactivity, volatility and interaction with components present in the salivary environment.
At the same time, the researchers observed that longer processing conditions favoured a greater presence of nitriles.
This suggests competition between different glucosinolate degradation pathways during the oral phase.
27 volatile compounds identified
Chemical analysis identified 27 volatile organic compounds produced during the simulated oral digestion of broccoli.
These compounds were mainly grouped into three families:
- isothiocyanates and thiocyanates;
- nitriles;
- sulfur-containing compounds.
Among the isothiocyanates, 2-phenylethyl isothiocyanate (PEITC) was the most abundant.
Compounds such as erucin were also identified, together with different nitriles and sulfur-containing compounds.
Some of these sulfur-containing compounds are also related to the characteristic aroma of broccoli and other cruciferous vegetables.
Figure 2. Comparison of the volatile organic compounds identified according to saliva proportion, contact time and mechanical processing time. Source: Gudino et al. (2026), LWT – Food Science and Technology.
The figure visually shows how different experimental conditions modify the resulting chemical profile.
Saliva proportion was the variable that produced some of the clearest differences.
Intermediate mixtures, especially 50% and 75%, were associated with a greater presence of several isothiocyanates, while the 100% saliva condition showed a profile more strongly associated with certain nitriles and sulfur-containing compounds.
Human saliva or artificial saliva?
One particularly interesting aspect of this study is the use of real human saliva.
Many experimental oral digestion models use artificial saliva.
These solutions can reproduce some important properties of saliva, such as pH, ionic strength or certain minerals. However, they cannot completely reproduce its biological complexity.
Human saliva contains a combination of:
water, proteins, enzymes, electrolytes and components related to the oral microbiota.
All of these elements can potentially interact with compounds present in food.
For this reason, considering saliva composition may be particularly important when the aim is to understand the release, transformation or bioaccessibility of molecules during the oral phase.
What was the most suitable condition?
Although the 50% saliva condition showed the highest mean total ITC value when considered independently, the researchers evaluated the different compounds and experimental parameters together.
The multivariate analysis showed that intermediate saliva mixtures, particularly 50% and 75%, were associated with the formation of ITCs such as erucin and PEITC.
Considering the overall balance between glucosinolate release and the formation of bioactive products, the authors selected the following condition:
75% saliva + 3 minutes of contact + 3 minutes of mechanical processing
as the optimised methodology for their experimental model.
Figure 3. Principal component analysis of the compounds and parameters studied during ex vivo oral digestion of broccoli. Source: Gudino et al. (2026), LWT – Food Science and Technology.
The PCA analysis reinforces the idea that oral-phase conditions can direct compounds towards different transformation pathways.
Intermediate saliva mixtures were associated with isothiocyanate formation, while other conditions showed stronger associations with nitriles or sulfur-containing compounds.
The oral phase is an active part of digestion
This study reinforces an important idea in food research:
the mouth is not simply the place where food is broken down before being swallowed.
Food structure, saliva quantity and composition, contact time and chewing can modify the compounds released during the earliest stages of digestion.
In addition, the authors themselves highlight an important limitation: the study used saliva from a single donor.
This raises a particularly relevant question for future research: to what extent could individual differences in saliva composition modify these transformations?
A better understanding of these interactions between saliva and the food matrix may help develop more physiologically relevant models and deepen our understanding of oral processing, bioaccessibility and inter-individual variability.
Reference
Gudino, I., Casquete, R., Benito, M. J., Martín, A., & Lamy, E. (2026). Influence of saliva and mechanical processing on the release of bioactive compounds during ex vivo oral digestion of broccoli. LWT – Food Science and Technology, 255, 119771.
