Can Lactoferrin, a Natural Mammalian Milk Protein, Assist in the Battle against COVID-19?

Notwithstanding mass vaccination against specific SARS-CoV-2 variants, there is still a demand for complementary nutritional intervention strategies to fight COVID-19. The bovine milk protein lactoferrin (LF) has attracted interest of nutraceutical, food and dairy industries for its numerous properties-ranging from anti-viral and anti-microbial to immunological-making it a potential functional ingredient in a wide variety of food applications to maintain health. Importantly, bovine LF was found to exert anti-viral activities against several types of viruses, including certain SARS-CoV-2 variants. LF's potential effect on COVID-19 patients has seen a rapid increase of in vitro and in vivo studies published, resulting in a model on how LF might play a role during different phases of SARS-CoV-2 infection. Aim of this narrative review is two-fold: (1) to highlight the most relevant findings concerning LF's anti-viral, anti-microbial, iron-binding, immunomodulatory, microbiota-modulatory and intestinal barrier properties that support health of the two most affected organs in COVID-19 patients (lungs and gut), and (2) to explore the possible underlying mechanisms governing its mode of action. Thanks to its potential effects on health, bovine LF can be considered a good candidate for nutritional interventions counteracting SARS-CoV-2 infection and related COVID-19 pathogenesis.

Correction to: Cheese consumption and risk of cardiovascular disease: a meta­analysis of prospective studies.

In the original publication, the funding and conflict of interest statements were not correct.

Cheese consumption and risk of cardiovascular disease: a meta-analysis of prospective studies.

PURPOSE: Cheese contains a high content of saturated fatty acids but also lists of potentially beneficial nutrients. How long-term cheese consumption affects the development of cardiovascular disease (CVD) is unclear. A meta-analysis of prospective observational studies was conducted to evaluate the risks of total CVD, coronary heart disease (CHD), and stroke associated with cheese consumption. METHODS: Potentially eligible studies were identified by searching PubMed and EMBASE databases and by carefully reviewing the bibliographies of retrieved publications and related reviews. The summary relative risks (RRs) with 95 % confidence intervals (CIs) were calculated using the random-effects model. RESULTS: The final analyses included 15 prospective studies. Most of the studies excluded prevalent CVD at baseline (14/15) and had a duration >10 years (13/15). The summary RR for high vs. low cheese consumption was 0.90 (95 % CI 0.82-0.99) for total CVD (7 studies, 8076 events), 0.86 (95 % CI 0.77-0.96) for CHD (8 studies, 7631 events), and 0.90 (95 % CI 0.84-0.97) for stroke (7 studies, 10,449 events), respectively. The restricted cubic model indicated evidence of nonlinear relationships between cheese consumption and risks of total CVD (P nonlinearity < 0.001) and stroke (P nonlinearity = 0.015), with the largest risk reductions observed at the consumption of approximately 40 g/d. CONCLUSIONS: This meta-analysis of prospective studies suggests a nonlinear inverse association between cheese consumption and risk of CVD.

6-methoxyflavanones as bitter taste receptor blockers for hTAS2R39.

Many (dietary) bitter compounds, e.g. flavonoids, activate bitter receptor hTAS2R39 in cell-based assays. Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4'-fluoro-6-methoxyflavanone, 6,3'-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 4'-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate.

Bitter taste receptor activation by flavonoids and isoflavonoids: modeled structural requirements for activation of hTAS2R14 and hTAS2R39.

Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 µM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure-activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the "bitter motif", introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

Evaluation of the bitter-masking potential of food proteins for EGCG by a cell-based human bitter taste receptor assay and binding studies.

Epigallocatechin gallate (EGCG) has been ascribed to several health benefits, but its bitter taste influences the liking of products with high concentrations of this compound. ß-Casein, in particular, and several gelatins are known as strong binders of EGCG, contrary to ß-lactoglobulin. The current study aimed at relating the EGCG-binding characteristics of those proteins and their food-grade equivalents to their effects on reducing bitter receptor activation by EGCG in vitro and their bitter-masking potential in vivo. Also in the bitter receptor assay, ß-casein showed the strongest effect, with a maximum reduction of hTAS2R39 activation of about 93%. A similar potency was observed for Na-caseinate. ß-Lactoglobulin had little effect on bitter receptor activation, as expected based on its low binding affinity for EGCG. The bitter-masking potential of Na-caseinate was confirmed in vivo using a trained sensory panel. ß-Lactoglobulin also slightly reduced EGCG bitter perception, which could not be directly related to its binding capacity. The bitter receptor assay appeared to be a valid tool to evaluate in vitro the efficacy of food proteins as complexing agents for masking bitterness.

Individually Modified Saliva Delivery Changes the Perceived Intensity of Saltiness and Sourness.

Individuals vary largely in their salivary flow and composition, and given the importance of saliva on perception of taste, this might influence how the tastant stimuli are perceived. We therefore hypothesise that altering the individual salivary flow rates has an impact on the perceived taste intensity. In this study, we investigated the role of saliva amount on the perceived taste intensity by excluding parotid saliva and adding artificial saliva close to the parotid duct at preset flow rates. Significant decreases in perception with increasing salivary flow rates were observed for citric acid and sodium chloride. This can partially be explained by a dilution effect which is in line with previous studies on detectable concentration differences. However, since the bitterness and sweetness remained unaffected by the salivary flow conditions and the dilution effect was comparable to that of saltiness, further explanation is needed. Furthermore, we investigated whether the suppression of taste intensity in binary mixtures (taste-taste interactions) could possibly be caused by the increased salivary flow rate induced by an additional taste attribute. The results show, however, that suppression of taste intensity in binary mixtures was not affected by the rate of salivation. This was more likely to be explained by psychophysics.

Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39.

The aim of this study was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 µM on hTAS2R14 and hTAS2R39 and EC(50) values of 29 and 49 µM, respectively. In addition, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar toward most isoflavonoid aglycones. By trend, threshold values were slightly lower on hTAS2R14. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely, glycitin, genistin, acetylgenistin, and malonylgenistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than fewer hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the determined bitter receptor threshold values, whereas those of other soy isoflavones are around or below their respective threshold value. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.

Temporal contrast of salt delivery in mouth increases salt perception.

The impact of salt delivery in mouth on salt perception was investigated. It was hypothesized that fast concentration changes in the delivery to the receptor can reduce sensory adaptation, leading to an increased taste perception. Saltiness ratings were scored by a panel over time during various stimulation conditions involving relative changes in NaCl concentration of 20% and 38%. Changes in salt delivery profile had similar effect on saltiness perception when delivered either by a sipwise method or by a gustometer. The impact of concentration variations and frequency of concentration changes was further investigated with the gustometer method. Five second boosts and 2 s pulses were delivered during 3 sequential 10-s intervals, whereas the delivered total salt content was the same for all conditions. Two second pulses were found to increase saltiness perception, but only when the pulses were delivered during the first seconds of stimulation. Results suggest that the frequency, timing, and concentration differences of salt stimuli can affect saltiness. Specifically, a short and intense stimulus can increase salt perception, possibly through a reduction of adaptation.

Branched chain aldehydes: production and breakdown pathways and relevance for flavour in foods.

Branched aldehydes, such as 2-methyl propanal and 2- and 3-methyl butanal, are important flavour compounds in many food products, both fermented and non-fermented (heat-treated) products. The production and degradation of these aldehydes from amino acids is described and reviewed extensively in literature. This paper reviews aspects influencing the formation of these aldehydes at the level of metabolic conversions, microbial and food composition. Special emphasis was on 3-methyl butanal and its presence in various food products. Knowledge gained about the generation pathways of these flavour compounds is essential for being able to control the formation of desired levels of these aldehydes.

Roasting effects on formation mechanisms of coffee brew melanoidins.

The effect of the roasting degree on coffee brew melanoidin properties and formation mechanisms was studied. Coffee brew fractions differing in molecular weight (Mw) were isolated from green and light-, medium-, and dark-roasted coffee beans. Isolated fractions were characterized for their melanoidin, nitrogen, protein, phenolic groups, chlorogenic acid, quinic acid, caffeic acid, and sugar content. It was found that the melanoidin level in all fractions correlated with both the nitrogen and the protein content. The melanoidin level also correlated with the phenolic groups' level and ester-linked quinic acid level. It was concluded that proteins and chlorogenic acids should be primarily involved in melanoidin formation. Initial roasting, from green to light-roasted beans, especially led to the formation of intermediate Mw (IMw) melanoidins when compared to high Mw (HMw) melanoidins. Indications were found that this IMw melanoidin formation is mainly due to Maillard reactions and chlorogenic acid incorporation reactions between chlorogenic acids, sucrose, and amino acids/protein fragments. Additionally, it was found that prolonged roasting predominantly led to formation melanoidins with a high Mw. Furthermore, arabinogalactans seem to be relatively more involved in melanoidin formation than galactomannans. It was hypothesized that chromophores may be formed or attached through the arabinose moiety of arabinogalactan proteins (AGP). Finally, it could be concluded that galactomannans are continuously incorporated in AGP-melanoidins upon roasting.

Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast.

The antioxidative properties of coffee brew fractions were studied using electron spin resonance spectroscopy using 2,2,6,6-tetramethyl-1-piperidin-1-oxyl (TEMPO) and Fremy's salt (nitrosodisulfonate) as stabilized radicals. TEMPO was scavenged by antioxidants formed during roasting and not by chlorogenic acid, whereas Fremy's salt was scavenged by all antioxidants tested including chlorogenic acid. The stabilized radical TEMPO allowed the exclusive measurement of roasting-induced antioxidants. The roasting-induced antioxidant activity of coffee brews increased with increasing degree of roast, and most of these antioxidants were formed during the initial roasting stage. The majority of these roasting-induced antioxidants were present in the high molecular weight fractions, indicating that the formation of these antioxidants preferably occurs at specific high molecular weight structures, likely being arabinogalactan and/or protein moieties which might be part of the melanoidin complex. It was found that chlorogenic acids most probably do not lose their antioxidant activity and phenolic characteristics upon incorporation in coffee melanoidins. The parameter fast reacting antioxidants (FRA) was introduced as an alternative for the antioxidative potential. FRA levels showed that coffee fractions rich in roasting-induced antioxidants exposed their antioxidant activity relatively slowly, which must be a consequence of its complex structure. Finally, the melanoidin content and the roasting-induced antioxidant activity showed a positive and linear correlation for the coffee brew fractions, showing that roasting-induced antioxidants are present within melanoidins. This is the first time that the formation of roasting-induced antioxidants could be directly correlated with the extent of Maillard reaction and melanoidin formation in a complex product such as coffee.

Low molecular weight melanoidins in coffee brew.

Analysis of low molecular weight (LMw) coffee brew melanoidins is challenging due to the presence of many non-melanoidin components that complicate analysis. This study focused on the isolation of LMw coffee brew melanoidins by separation of melanoidins from non-melanoidin components that are present in LMw coffee brew material. LMw coffee fractions differing in polarity were obtained by reversed-phase solid phase extraction and their melanoidin, sugar, nitrogen, caffeine, trigonelline, 5-caffeoylquinic acid, quinic acid, caffeic acid, and phenolic groups contents were determined. The sugar composition, the charge properties, and the absorbance at various wavelengths were investigated as well. The majority of the LMw melanoidins were found to have an apolar character, whereas most non-melanoidins have a polar character. The three isolated melanoidin-rich fractions represented 56% of the LMw coffee melanoidins and were free from non-melanoidin components. Spectroscopic analysis revealed that the melanoidins isolated showed similar features as high molecular weight coffee melanoidins. All three melanoidin fractions contained approximately 3% nitrogen, indicating the presence of incorporated amino acids or proteins. Surprisingly, glucose was the main sugar present in these melanoidins, and it was reasoned that sucrose is the most likely source for this glucose within the melanoidin structure. It was also found that LMw melanoidins exposed a negative charge, and this negative charge was inversely proportional to the apolar character of the melanoidins. Phenolic group levels as high as 47% were found, which could be explained by the incorporation of chlorogenic acids in these melanoidins.

Incorporation of chlorogenic acids in coffee brew melanoidins.

The incorporation of chlorogenic acids (CGAs) and their subunits quinic and caffeic acids (QA and CA) in coffee brew melanoidins was studied. Fractions with different molecular weights, ionic charges, and ethanol solubilities were isolated from coffee brew. Fractions were saponified, and the released QA and CA were quantified. For all melanoidin fractions, it was found that more QA than CA was released. QA levels correlated with melanoidin levels, indicating that QA is incorporated in melanoidins. The QA level was correlated with increasing ionic charge of the melanoidin populations, suggesting that QA may contribute to the negative charge and consequently is, most likely, not linked via its carboxyl group. The QA level correlated with the phenolic acid group level, as determined by Folin-Ciocalteu, indicating that QA was incorporated to a similar extent as the polyphenolic moiety from CGA. The QA and CA released from brew fractions by enzymes confirmed the incorporation of intact CGAs. Intact CGAs are proposed to be incorporated in melanoidins upon roasting via CA through mainly nonester linkages. This complex can be written as Mel=CA-QA, in which Mel represents the melanoidin backbone, =CA represents CA nonester-linked to the melanoidin backbone, and -QA represents QA ester-linked to CA. Additionally, a total of 12% of QA was identified in coffee brew, whereas only 6% was reported in the literature so far. The relevance of the additional QA on coffee brew stability is discussed.

Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins.

The charge properties of melanoidins in high molecular weight (HMw) coffee brew fractions, isolated by diafiltration and membrane dialysis, were studied. Ion exchange chromatography experiments with the HMw fractions showed that coffee brew melanoidins were negatively charged whereas these molecules did not expose any positive charge at the pH of coffee brew. Fractions with different ionic charges were isolated and subsequently characterized by means of the specific extinction coefficient (K(mix 405nm)), sugar composition, phenolic group content, nitrogen content, and the arabinogalactan protein (AGP) specific Yariv gel-diffusion assay. The isolated fractions were different in composition and AGP was found to be present in one of the HMw fractions. The AGP accounted for 6% of the coffee brew dry matter and had a moderate negative charge, probably caused by the presence of uronic acids. As the fraction that precipitated with Yariv was brown (K(mix 405nm) = 1.2), compared to a white color in the green bean, it was concluded that these AGPs had undergone Maillard reaction resulting in an AGP-melanoidin complex. The presence of mannose (presumably from galactomannan) indicates the incorporation of galactomannans in the AGP-melanoidin complex. As the uronic acid content in the more negatively charged melanoidin-rich, AGP-poor HMw fractions decreased, it was hypothesized that acidic groups are formed or incorporated during melanoidin formation.

High molecular weight melanoidins from coffee brew.

The composition of high molecular weight (HMw) coffee melanoidin populations, obtained after ethanol precipitation, was studied. The specific extinction coefficient (K(mix)) at 280, 325, 405 nm, sugar composition, phenolic group content, nitrogen content, amino acid composition, and non-protein nitrogen (NPN) content were investigated. Results show that most HMw coffee melanoidins are soluble at high ethanol concentrations. The amino acid composition of the HMw fractions was similar, while 17% (w/w) of the nitrogen was NPN, probably originating from degraded amino acids/proteins and now part of melanoidins. A strong correlation between the melanoidin content, the NPN, and protein content was found. It was concluded that proteins are incorporated into the melanoidins and that the degree of chemical modification, for example, by phenolic groups, determines the solubility of melanoidins in ethanol. Although the existence of covalent interaction between melanoidins and polysaccharides were not proven in this study, the findings suggest that especially arabinogalactan is likely involved in melanoidin formation. Finally, phenolic groups were present in the HMw fraction of coffee, and a correlation was found with the melanoidin concentration.

Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models.

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products.

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.

Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation.

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.

New device to simulate swallowing and in vivo aroma release in the throat from liquid and semiliquid food systems.

This paper describes a novel device to simulate in vivo aroma release from liquids. This artificial throat simulates the act of swallowing followed by exhalation and shows aroma release curves that are similar in shape to in vivo release profiles. Liquids are poured down a tube, and a thin liquid film remains at the inner wall of the tube. Subsequently, aroma compounds release from this film into a stream of air flowing through this tube, which is analyzed by MS-Nose analysis. The effects of air flow rate, contact time with glass surface, presence of saliva, and addition of whey protein, as well as volume, concentration, temperature, and viscosity of the liquid have been studied and compared with aroma release measurements in vivo. A high level of agreement was found. These results confirm the importance of swallowing for aroma release of liquids, as mentioned in the literature, and the usefulness of the new mimicking device.