# Effect of Different Ratios of Cow Manure and Chemical Fertilizers on Fruit Quality of Gala Apples
## 2 Materials and Methods
### Physical Chemical Analysis, Organic Acids, and Sugar Fraction  
A total of 10 apple fruits from each treatment were selected to determine colour, size, total soluble solids (TSS), total titratable acidity (TA), and weight. TSS were determined by digital refracometer (PLA-1, Japan); total titratable acids and vitamin C (VC) were determined by 905 automatic potentiometric titrator. The fruit surface colour of samples was measured using a precision handheld colourimeter (NR20 XE, China). Fruit surface colour mainly consisted of five indicators: the L\({}^{*}\) value represents the brightness of the colour, and the value range is from 0 to 100, whereby 100 represents white and 0 represents black; a* represents the red and green degree, and the value range is from \(-\)60 to \(+\)60; a* is red and purple when positive, and green and blue when negative, and the larger the absolute value is, the darker the colour is; b* represents the yellow and blue degree, and the value range is also from \(-\)60 to \(+\)60; so, 42 is yellow when positive, and blue when negative, and the larger the absolute value is, the darker the colour is. From the a* and b* values, C* (Chroma) and H\({}^{0}\) (hue angle) can be calculated. Where C* = [a\({}^{*2}\) + b\({}^{*2}\)]1/2 indicates the colour saturation and H\({}^{0}\) is the hue angle. When a* > 0 and b* > 0, H\({}^{0}\) = tan\({}^{-1}\) (b*/a*); when a* < 0 and b* > 0, H\({}^{0}\) = 180 + tan\({}^{-1}\) (b*/a*); when h0 = 0 indicates red, H\({}^{0}\) = 90\({}^{\circ}\) indicates yellow, and H\({}^{0}\) = 180\({}^{\circ}\) indicates green [27].  
\begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Treatment**} & \multicolumn{8}{c}{**2017–2018**} & \multicolumn{8}{c}{**2019–2020**} \\  & **N** & **P\({}_{2}\)O\({}_{3}\)** & **K\({}_{2}\)O** &
\begin{tabular}{c} **Cow** \\ **Mature (N)** \\ \end{tabular} & **Total N** & **N** & **P\({}_{2}\)O\({}_{3}\)** & **K\({}_{2}\)O** & **Cow manure (N)** & **Total N** \\ \hline CK & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 \\
100\% CF & 240 & 120 & 240 & 0 & 240 & 480 & 240 & 480 & 0 & 480 \\
75\% CF + 25\% CM & 180 & 90 & 180 & 60 & 240 & 360 & 180 & 360 & 120 & 480 \\
50\% CF + 50\% CM & 120 & 60 & 120 & 120 & 240 & 240 & 120 & 240 & 480 \\
25\% CF + 75\% CM & 60 & 30 & 60 & 180 & 240 & 120 & 60 & 120 & 360 & 480 \\
100\% CM & 0 & 0 & 0 & 240 & 240 & 0 & 0 & 0 & 480 & 480 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Nitrogen, phosphorus, potassium and organic fertilizer inputs for different treatments (kg/hm\({}^{2}\)).  
Organic acids and sugars were identified and quantified according to [PERSON] et al. [28] with some modifications. The apple juices were prepared by hand-squeezing in a commercial juicer. Then, the juices were centrifuged at 12,000\(\times\)\(g\) for 30 min (HITACHI RXII, Japan), filtered through a 0.45 \(\upmu\)m Millipore filter, and injected (1.5 mL) into a high-performance liquid chromatograph (HPLC) system. Organic acids were separated on a Supelcopgel C-610H column (30 cm \(\times\) 7.8 mm) fitted with a Supelcopguard column (2.5 cm \(\times\) 4 mm) and a diode array detector (210 nm) was used for the detection. For the determination of sugars, the same HPLC system and conditions were used, but detection was performed with a refractive index detector. Standard curves of pure organic acids and sugars were used for quantification. The concentrations of organic acids and sugars are expressed in mg/g and %, respectively.