L-Lactic Acid

In the behavioural room in The Netherlands and in a semi-field screenhouse in Kenya, similar experimental setups were used to carry out a dual-choice experiment comparing mosquito trapping efficacy of the Suna trap against the MM-X trap (see Additional file 1). During any given replicate of the experiment, one Suna trap and one MM-X trap were suspended simultaneously in opposite corners of either the behavioural room or the screenhouse. Suna traps were suspended at 30 cm above the ground, as demonstrated to be the most effective height for this trap during field experiments. MM-X traps were suspended at 15 cm above ground level [29 (link)]. The positions of the traps were alternated for each experimental replicate and mosquitoes were released from a point equidistant from the two traps. In The Netherlands and Kenya, both traps were baited with a blend of ammonia, L-lactic acid, tetradecanoic acid, 3-methyl-1-butanol and 1-butylamine [38 ]. In the behavioural room, pressurized CO₂ was supplied from a cylinder at 250 cc/min. In the semi-field setup, CO₂ was produced through a yeast and molasses fermentation process (250 ml molasses, 17.5 g yeast, 2 litres water), shaken vigorously for 30 seconds [39 (link)].
In The Netherlands, 50 unfed female An. coluzzii were used for each experimental replicate and experiments were carried out during the mosquito dark photoperiod (artificially set between 00:00 h and 12:00 h) with a duration of one hour per replicate. In Kenya, 200 unfed female An. gambiae were released into the screenhouse at 20:00 h and the experiment was stopped at 06:30 h the following morning.
At the end of all laboratory and semi-field experiments traps were placed in a freezer at -20°C in order to knock down mosquitoes for counting. Temperature and relative humidity were measured during all experimental replicates using a Tinytag^® Ultra data logger (model TGU-1500, INTAB Benelux, The Netherlands). Any remaining mosquitoes died during the day because of exposure to high daytime temperatures and starvation.

Mosquito Magnet® X (MM-X) traps [29 (link),30 (link)] were baited with CO₂ and a five-compound odour blend, which simulates the smell of a human foot [18 (link),28 ]. The individual compounds of the attractive blend were released from nylon strips (cut from panty hoses: 90% polyamide, 10% spandex, Marie Claire®) [31 (link)]. Concentrations were optimised for this set-up and release method: ammonia (2.5% in water), L-(+)-lactic-acid (85%), tetradecanoic acid (0.00025 g/l in ethanol), 3-methyl-1-butanol (0.000001% in water) and butan-1-amine (0.001% in paraffin oil) (see Table 3). Nylon strips (26.5 cm × 1 cm) were impregnated with the attractive compounds by dipping three strips in 3.0 ml of compound in a 4 ml screw top vial (Experiment 1) or by dipping individual strips into an Eppendorf tube containing 1 ml of solution (Experiment 2). Before use, strips were dried for 9–10 h at room temperature. During experiment 1 for every experimental night a set of freshly impregnated strips was used. During experiment 2 strips were used for a maximum of 12 consecutive nights. During daytime, the strips were packed in aluminium foil and stored at 4°C in a refrigerator.
The five strips were held together with a safety pin and hung in the outflow opening of the MM-X trap using a plastic covered clip. CO₂ was produced by mixing 17.5 g yeast with 250 g sugar and 2.5 L water [32 (link)] and released from the MM-X trap together with the odours. MM-X traps equipped with the attractive blend were positioned with the outflow opening at the optimal height of 15–20 cm above the floor surface [33 (link)].

The blend was developed through a sequential procedure (Fig. 1), starting with a weakly attractive primary mixture consisting of 2.5% ammonia solution and 500 ml/min of CO₂ gas. Onto this primary mixture, L-lactic acid was first added followed by the other aliphatic carboxylic acids initially one at a time to determine their optimal concentrations, and then jointly at their optimal concentrations to create the final blend. Whenever each of the candidate compounds were added, we iteratively varied its concentrations until the point when the resulting mixture was maximally competitive, in terms of its attractiveness to laboratory reared mosquitoes relative to the original mixture. This final concentration was considered the optimum for each respective candidate compound.
L-Lactic acid was the first to be added onto the primary mixture. The treatment trap was baited with the primary mixture plus different concentrations of L-lactic acid. The control trap on the other hand was baited with only the primary mixture. For each concentration of L-lactic acid, four replicates were conducted each lasting six hours, and between which we rotated the positions of the traps so as to minimize directional bias. The most significant improvement in attractiveness was determined to occur when L-lactic acid was added at 85% concentration (which was the undiluted formulation of L-lactic acid as purchased from the manufacturer).
Previously, the synergistic effect of L-lactic acid when combined with ammonia and a blend of carboxylic acids, has been demonstrated [17] (link), [18] (link). Therefore in the rest of our assays, all other aliphatic carboxylic acids were all tested in combination with 85% L-lactic acid, each time comparing the resulting mixture with the original mixture as before. In the case of these other carboxylic acids, the treatment trap was therefore baited with: 1) the primary mixture, 2) undiluted L-lactic acid and 3) an iteratively selected concentration of a selected carboxylic acid, while the control trap was baited with only the primary mixture. For each carboxylic acid, the optimal concentration was determined by iterating until the point when the resulting mixture was maximally attractive relative to the primary mixture.
After the optimal concentrations for all the carboxylic acids had been determined, all the compounds were added to the primary mixture at those respective concentrations to form the final synthetic odor blend. The synthetic blend therefore consisted of CO₂ gas flowing at 500 ml/min plus hydrous solutions of ammonia (2.5%), L-lactic acid (85%), and the other aliphatic carboxylic acids: propionic acid (C3) at 0.1%, butanoic acid (C4) at 1%, pentanoic acid (C5) at 0.01%, 3-methylbutanoic acid (3mC4) at 0.001%, heptanoic acid (C7) at 0.01%, octanoic acid (C8) at 0.01% and tetradecanoic acid (C14) at 0.01%. Finally, a variant of the blend was formulated by removing the unpleasant smelling 3mC4 in an attempt to improve the appeal of the blend to potential users.

The concentrations of citric acid, fumaric acid, gluconic acid, glucuronic acid, hippuric acid, lactic acid, maleic acid, malic acid, malonic acid, orotic acid, oxalic acid, pyruvic acid, succinic acid, and uric acid were quantified with external calibration, in triplicate, by UPLC-MS/MS, using an Acquity system equipped with an HSS T3 column (Waters), as described previously (De Bruyn et al., 2017 (link)), except that eluent A contained 2% (v/v) methanol (Merck). Samples were prepared by addition of 700 μL of a 1:1 (v/v) mixture of ultrapure water and methanol (Merck), containing 20 mg/L of salicylic acid (Fluka, Buchs, Switzerland) as IS, to 100 μl of the aqueous extracts, followed by microcentrifugation at 18,000 × g for 15 min, and filtering with a 0.2-μm LG H-PTFE filter (Millex; Merck) before injection (2 μL) into the column.
The ratio between D-lactic acid and L-lactic acid was quantified, in duplicate, by UPLC-MS/MS, using an Acquity system equipped with an Astec Chirobiotic column (Supelco; Bellefonte, PA, United States). The mobile phase consisted of an isocratic flow of 15% ultrapure water with 33.3 mM ammonium acetate (VWR International) and 85% acetonitrile (Merck) at a constant flow rate of 0.6 mL/min. Samples were prepared by addition of 100 μL of aqueous extract to 900 μL of a solution containing 85% acetonitrile (Merck), 15% ultrapure water, 0.386 g/L of ammonium acetate (VWR International), and 4.0 mg/L of salicylic acid (IS; Fluka). The mixtures were vortexed for 5 min, followed by microcentrifugation at 18,000 × g for 15 min, and filtering with a 0.2-μm LG H-PTFE filter (Millex; Merck) before injection (10 μL) into the column.

Microbiological analyses were performed in triplicate at the sampling points described in Figure 1: on day 0 (just after stuffing), during the production (before changing processing temperature on day 2, 7, 16 and 23 for FT and day 3, 14 and 21 for ST) and after storage. In total, 138 data points distributed all along the challenge test were obtained.
Product a_w was measured with an AquaLab^TM Series 3TE instrument (Decagon Devices Inc., Pullman, WA, USA). The pH was determined with a penetration probe (PH25 pHmeter) and 52–32 electrode (Crison Instrument SA, Alella, Spain). Lactic acid (D- and L- lactic acid, in g/100 g) was quantified with the D-/L-Lactic Acid (D-/L-Lactate) Assay kit (Megazyme International, Wicklow, Ireland) according to manufacturer instructions.
To enumerate LAB and Salmonella, 15 g of chopped product was ten-fold w/v diluted and homogenized in saline solution (0.85% NaCl and 0.1% Bacto Peptone) for 60 s in a Smasher^® (bioMérieux, Marcy-l’Étoile, France). This initial dilution was subsequently 10-fold serially diluted in saline solution. LAB counts were determined in Man-Rogosa-Sharpe (MRS) agar plates (Merck, Darmstadt, Germany) anaerobically incubated for 72 h at 30 °C in sealed jars with AnaeroGen sachet (Oxoid Ltd.) [29 ]. Salmonella was enumerated on chromogenic agar (CHROMagar^TM Salmonella Plus; Scharlab, Spain) incubated for 48 h at 37 °C [5 (link)]. Samples with expected Salmonella counts below the quantification limit (<10 cfu/g), were enriched in TSBYE at 37 °C for 48 h and the detection/non-detection of the pathogen was determined by plating on CHROMagar^TM Salmonella Plus.