Bag mixer 100 minimix

For the isolation of AAB, six date vinegar samples were prepared in the laboratory. A low-quality date (Phoenix dactylifera L.) variety ‘Um al Sila’ was used for this purpose and it was collected from a local farm in Al-Suwaiq, Al-Batinah Governorate, Oman. The samples were mixed with sterile distilled water in a ratio of 1:4 (w/v), 175 g date fruit and 525 mL distilled water, in a stomacher bag and homogenized using a stomacher (Bagmixer 100 MiniMix, Interscience, Bois Arpents, France) for 1 min. After that, date broths were sieved into conical flasks and closed with sterile cotton plugs. All samples were incubated at 30 °C in an incubator (Gallen Kamp, Cambridge, UK) statically to permit spontaneous fermentation.

Samples were collected at the end of the aerated basins at the Rya WWTP, a WWTP treating both industrial and municipal wastewater [28 (link)]. Permission to enter the Rya WWTP and to collect activated sludge samples was granted by Gryaab AB (owner and operator of the WWTP). 50 mL of sample were centrifuged at 4000xg for 3 minutes and the resulting pellet was stored at -20°C within 1.5 h from collection. DNA was extracted using the Power Soil DNA Extraction Kit (MoBio Laboratories). The frozen sludge pellets were thawed, 15 mL sterile water were added and the samples were homogenized by 6 min of mixing in a BagMixer 100 MiniMix (Interscience). Water was removed by centrifugation (4000xg for 3 minutes) and DNA was extracted from 0.25 g of homogenized sludge pellet according to the manufacturer’s instructions.

The determination was performed based on the method described by SR ISO 21527-2/2008 standard [35 ], by Antoniewska et al., (2018) [36 (link)] and Naghy et al., (2017) [37 (link)]. Briefly, 5 g of each sample were mixed with 45 mL of 0.1% peptone water diluent (Oxoid Ltd., Basingstoke, Hampshire, England) in a stomacher (Bag Mixer 100 MiniMix, Interscience, St. Nom, France). Further decimal dilution (10⁻²) was prepared in 0.1% peptone water diluent. All dilutions were inoculated in duplicate. 100 µL of the initial dilution (10⁻¹) were aseptically transferred to a Petri dish containing Dichloran-Glycerol (DG18) agar (Oxoid Ltd., Basingstoke, Hampshire, England) using a sterile pipette and spread immediately with a Drigalski-spatula.
The procedure was repeated with further decimal dilution (10⁻²). The inoculated dishes were inverted and incubated at 25 °C for 5 days. After incubation, the visible colonies on selected plates (less than 150 colonies) were counted using a colony counter Colony Star 8500, (Funke-Dr. N. Gerber Labortechnik, Berlin, Germany).

Samples were analyzed in a safety cabinet (Purifier class II, Labconco, Kansas, USA) and cut using sterile scalpels. Some fruits (banana, mango, papaya, pomegranate, and watermelon) were peeled and the inside flesh was analyzed. Twenty-five grams of the cut sample was weighed in a sterile stomacher bag and mixed with 225 mL of Maximum Recovery Diluent (MRD) and homogenized for 1 min using a stomacher (Bagmixer 100 MiniMix, Interscience, Bois Arpents, France). Serial dilutions were prepared from the original homogenate in MRD. Aerobic plate count (APC) was performed by spread plate method. The plates of standard plate count agar (SPCA) were incubated at 35°C for 48 hrs [7 (link)]. The count of Enterobacteriaceae was performed on Violet Red Bile Glucose (VRBG) agar by pour plate method and incubation at 35°C for 24 hrs. E. coli was counted on Tryptone Bile X-glucuronide (TBX) medium and the plates were incubated at 2 temperatures: 30 and 44°C for 24 hrs [14 (link)]. Staphylococcus aureus was counted on Baird-Parker (BP) agar after incubation at 35°C for 24 hrs [7 (link)]. Enterococcus was counted on Slanetz agar after incubation at 35°C for 48 hrs [15 (link)]. All microbiological media were from Oxoid, England, and all experiments were repeated three times.