## 2 Materials and Methods
### Concept of Volumetric Zone of Inhibition and Calculation  
The concept of volumetric zone of inhibition was developed using Equation (1):  
\[A=\pi r^{2}\;(r\;=\;D/2);\;V=A\cdot H\]  
where \(H\), \(A\), \(V\) represent the thickness, area and the volume covered by the grape pomace extract agar, respectively. Prior to determining this volume, \(A=\pi r^{2}\;(r\;=\;D/2)\) was used to calculate the area covered by the grape pomace extract agar (GPE) agar, where \(r\) and \(D\) represent the radius, and the diameter of the petri dish, respectively.  
The data obtained from the measured zone of inhibition, the thickness of the GPE agar and the diameter of the pierced wells on the GPE agar were used in Equation (1). The diameter zone of inhibition (\(D\;=\;D_{o}-d\)) measured around the well (Figure 1) is a resultant of the volume of 20 \(\mu\)L of the biopreservation compounds used (BCU) which was introduced in the pierced well.  
This volume (20 \(\mu\)L) of the inoculated, solidified GPE agar was the concept basis of the volumetric zone of inhibition (VZI). Since 1 cm\({}^{3}\) = 1 mL = 10\({}^{-3}\) L, the calculated volume (\(V=A\cdot H\)) units expressed in liters (L) was used for consistency. In this study, the VZI interpretation was based on the fact that 20 \(\mu\)L (0.02 mL) of biopreservation compounds sample was sufficient to inhibit the growth of the spoilage organism inoculated at 1 \(\times\) 10\({}^{6}\) CFU mL\({}^{-1}\) in a defined volume (L) of the GPE agar plate. The term inhibitory activity (L mL\({}^{-1}\)) was then adopted to describe the growth inhibitory effects which are reported as the volume of contaminated (inoculated) solidified media (L CSM) per mL of biopreservation compounds used (mL BCU). The units were presented as L CSM mL\({}^{-1}\) BCU.  
Figure 1: Outline of the concept developed to calculate the volumetric zone of inhibition (VZI) (adapted from [PERSON] et al., 2017 [23]).  
Preliminary Assessments for Biopreservation Compounds Production, Chemical Analysis and Growth Inhibition Assay  
The preliminary assessment for production of biopreservation compounds was done using Chenin Blanc GPE. A concentration of 10\({}^{6}\) cells mL\({}^{-1}\) of _C. pyralidae_ Y1117, _P. kluyveri_ Y1125 and _P. kluyveri_ Y1164 were inoculated separately (in triplicate) in 250 mL Erlenmeyer flasks, containing 150 mL of the autoclaved GPE broth at a concentration of 100, 150 and 200 g L\({}^{-1}\). The flasks were incubated for 32 h at 25 \({}^{\circ}\)C in a shaking incubator (LM-53 OR, RKC(r) Instrument INC, Ohta-ku Tokyo, Japan) set at 150\(\times\) rpm. Samples were withdrawn every 4 h, centrifuged at 5000\(\times\) rpm for 5 min and the supernatants were filtered using 0.22 \(\upmu\)m sterile nylon membrane filters. Filtered samples (1 mL) were analyzed for total sugar and YAN while the remaining aliquots were used for the growth inhibition assay.  
The growth inhibition assay was adapted from [10] with some modifications. The GPE agar used for growth inhibition assay was prepared by supplementing the GPE broth (150 g\(\cdot\)L\({}^{-1}\)) with 2% agar bacteriological (Biolab, Merck, South Africa).  
A well with a 5-mm diameter was drilled on GPE agar plates using an agar driller. Prior to drilling, the GPE agar plates were seeded with 10\({}^{6}\) cells mL\({}^{-1}\) of either _Z. bailli, D. bruxellensis_ or _D. anomala_[10]. In the agar wells, a volume of 20 \(\upmu\)L of the crude biopreservation compounds was spotted, subsequent to incubation at 22 \({}^{\circ}\)C until clear zones of inhibition were observed around the 5 mm wells. The plates were then assessed for biopreservation activity, which was quantified as described by [PERSON] et al. [23].