## 3 Results and Discussion
### S. aureus Challenge Test  
Lower inoculant can be used to precisely indicate the capacity of a product to support microbial growth. In this study, a \(5\times 5\times 2\) cm\({}^{3}\) sample was cut from the center of the loaf after it had cooled, and the challenge test was performed by inoculating 0.5 wt. % culture with 2-3 log CFU g\({}^{-1}\)_S. aureus_. Because _S. aureus_ produces enterotoxins at levels above 6 log CFU g\({}^{-1}\)[23], the outcome of the challenge test is based on a threshold of 6 log CFU g\({}^{-1}\). The results are shown in Figure 4. Because discernible mold had appeared on the bread from the sixth day onwards, the observation period ended on the fifth day. The initial _S. aureus_ counts inoculated on all groups of bread on the first day were within 2.62-2.70 log CFU g\({}^{-1}\), and no significant differences were observed on the second day of _S. aureus_ inoculation as this was the lag phase. The _S. aureus_ counts of the control, 2%, 4%, 6%, and 8% SPI-fortified breads were 3.28 log CFU g\({}^{-1}\), 3.62 log CFU g\({}^{-1}\), 3.55 log CFU g\({}^{-1}\), 3.44 log CFU g\({}^{-1}\), and 3.68 log CFU g\({}^{-1}\), respectively. On the third day, the _S. aureus_ count of the control (3.90 log CFU g\({}^{-1}\)) was slightly lower than those of the 2%, 4%, 6%, and 8% SPI-fortified breads (4.50 log CFU g\({}^{-1}\), 4.44 log CFU g\({}^{-1}\), 4.21 log CFU g\({}^{-1}\), and 4.45 log CFU g\({}^{-1}\), respectively.). On the fourth day, the _S. aureus_ counts of the control, 2%, 4%, 6%, and 8% SPI-fortified breads were 4.38 log CFU g\({}^{-1}\), 5.44 log CFU g\({}^{-1}\), 5.44 log CFU g\({}^{-1}\), 5.25 log CFU g\({}^{-1}\), and 5.37 log CFU g\({}^{-1}\), respectively. This shows that the SPI-fortified breads had a significantly higher _S. aureus_ count than the control (\(p<0.05\)). On the fifth day, the _S. aureus_ counts of the control, 2%, 4%, and 6% SPI-fortified breads were 5.12 log CFU g\({}^{-1}\), 5.79 log CFU g\({}^{-1}\), 5.81 log CFU g\({}^{-1}\), and 5.63 log CFU g\({}^{-1}\), respectively, while that of the 8% SPI-fortified bread had risen to 6.58 log CFU g\({}^{-1}\). Figure 3: Effects of partially substituting high-gluten flour with soy protein isolate on the mold and yeast growth in bread. According to the challenge test results, the _S. aureus_ count was below 6 log CFU g\({}^{-1}\) within the first four days of storage and had successfully passed the challenge test. The control bread passed the test within the first five days of storage. A possible reason for this difference is the amino acid composition of SPI in the SPI-fortified and control breads, as well as the difference between their pH values. Comparing SPI and flour protein composition, SPI contains 6.14% lysine and 2.47% methionine and cysteine, while flour protein contains 1.99% lysine and 4.52% methionine and cysteine [58; 59]. Hence, a more balanced intake of essential amino acids can be achieved by eating bread made from these two different sources of protein. Leucine, isoleucine, and valine are branched-chain amino acids that provide environments that are conducive to _S. aureus_ protein and branched-chain fatty acid formation. [PERSON] et al. [60] found a positive correlation between _S. aureus_ growth and leucine and isoleucine levels in composite milk. The branched-chain amino acids in SPI account for 16.08% of all amino acids, while flour protein contains 13.69% branched-chain amino acids [58; 59]. This could be a reason why SPI-fortified bread is more conducive to _S. aureus_ growth than the control. As shown in Figure 2C, the bread pH increases with SPI use. [PERSON] et al. [26] reported that lowering the dough pH prolongs the lag phase of _S. aureus_ growth and shortens the microbial growth rate. [PERSON] et al. [61] studied the effects of pH on the biofilm formation of _S. aureus_ and found a positive correlation between pH and _S. aureus_ growth. The _S. aureus_ count exceeded 5 log CFU g\({}^{-1}\) on the fourth day. Table 4 shows the effect of SPI on the generation time and the reproductive rate of _S.