Fermentation characteristics and post-acidification of yogurt by Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 at optimal inoculum ratio

This study aimed to investigate the symbiosis be-tween Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047. In addition, the effect of their different inoculum ratios was determined, and comparison experiments of fermentation characteristics and storage stability of milk fermented by their mono-cultures and cocultures at optimal inoculum ratio were performed. We found the time to obtain pH 4.6 ( t pH4.6 ) and ∆pH during storage varied among 6 inoculum ratios (1:1, 2:1, 10:1, 19:1, 50:1, 100:1). By the statistical model to evaluate the optimal ratio, the ratio of 19:1 was selected which exhibited high acidification rate and low post-acidification with pH values remaining 4.2 – 4.4 after a 50-d storage. Among the 3 groups included in our analyses, i.e., the mono-cultures of S. thermophilus CICC 6038 (St) and Lb . bulgaricus CICC 6047 (Lb) and their cocultures (St+Lb) at 19:1, the coculture group showed higher acidification activity, improved rheological properties, richer typical volatile compounds, more desirable sensor quality after the fermentation process than the other 2 groups. However, the continuous accumulation of acetic acid during storage showed that acetic acid was more highly correlated with post-acidification than D-lactic acid for the Lb group and St+Lb group. Our study emphasized the importance of selecting an appropriate bacterial consortium at the optimal inoculum ratio to achieve favorable fermentation performance and enhanced post-acidification stability during storage.


INTRODUCTION
Streptococcus thermophilus and Lactobacillus delbrueckii ssp.bulgaricus are homofermentative lactic acid bacteria (LAB) that play a crucial role in the production of yogurt products worldwide.The mixed culture of S. thermophilus and Lb.bulgaricus is a classic example of a synthetic microbial consortium with clear links to product quality in the food fermentation industry (Eddy J Smid et al., 2013).These 2 LAB species share the same ecological niche and engage in symbiotic or competitive relationships, known as proto-cooperation, by exchanging metabolites and signaling molecules (Enuo Liu et al., 2016;Pascal Courtin and Françoise Rul, 2004).Typically, S. thermophilus is thought to provide formic acid, folic acid, carbon dioxide (CO 2 ), and fatty acids to initiate the growth of Lb. bulgaricus, whereas Lb. bulgaricus is assumed to produce a surplus of peptides and free amino acids to meet the biosynthetic demands of S. thermophilus (Luciana Herve-Jimenez et al., 2009;Sander Sieuwerts et al., 2010).In addition, these strains can also compete for environmental nitrogen utilization, and their metabolites may negatively affect each other during fermentation.Thus, proto-cooperation is a crucial determinant of the fermentation process and the final quality of yogurt products (Sarn Settachaimongkon et al., 2014).
The specific microbial strains and environmental conditions significantly affect the nature and strength of microbial interactions within the yogurt consortium (Sarn Settachaimongkon et al., 2014).Previous studies have showed that efficient symbiosis between the bacteria can boost mutual growth, accelerate acidification speed, increase exopolysaccharides (EPS) synthesis, and improve texture and flavor formation (Francoise Rul, 2017).However, the complexity and diversity of these interactions remain unknown.Recent studies have examined the effects of factors such as urease (R. Yamauchi et al., 2019), NADH oxidase (Hiroshi Horiuchi et al., 2014), glutathione synthetase (Ting Wang Fermentation characteristics and post-acidification of yogurt by Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp.bulgaricus CICC 6047 at optimal inoculum ratio Yuanyuan Ge,12 Xuejian Yu,2 Xiaoxin Zhao, 2 Chong Liu, 2 Ting Li, 2 Shuaicheng Mu, 2 Lu Zhang, 2 Zhuoran Chen,1 Zhe Zhang, 2 Zhiquan Song, 2 Hongfei Zhao, 1 Su Yao, 2 * and Bolin Zhang 1 * Xu et al., 2016), and fumaric acid (Eri Yamamoto et al., 2021) on the symbiosis relationship between S. thermophilus and Lb.bulgaricus, however, these studies have been limited to the most extensively used starter strains.Furthermore, the effect of different inoculum ratios of yogurt bacteria on the fermentation characteristics has garnered industrial interest (Tong Dan et al., 2023), as reported in the study showing obvious differences in volatile flavor compounds profiles among 6 different proportional combinations (Tong Dan et al., 2017).Therefore, to achieve desired properties, such as strong fermentation capability, distinctive flavors, low post-acidification, and probiotic effects, screening more combinations of starter cultures with improved synergistic effects, and determining their optimal proportions, have become crucial in yogurt manufacturing practices.
Post-acidification, resulting from the ongoing accumulation of organic acids in starter cultures at low temperatures, compromises product stability and poses a significant challenge in yogurt production.The primary lactate product by L. delbrueckii ssp.bulgaricus, D-lactic acid, has been identified as the main contributor to this issue and is implicated in certain pathologies upon accumulation (Herrero et al., 2004;Maria et al., 2016).Thus, it is expected that D-lactic acid could be restrained in yogurt fermentation process (Jifeng et al., 2015).In response to this challenge, various approaches have been developed to combat post-acidification (Gaurav Kr Deshwal et al., 2021), with the selection of suitable strain combinations being identified as the safest and most effective solution.This solution involves choosing strains with weak post-acidification capacity while maintaining the desirable technological performance to ensure high product quality.
S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047, isolated from traditional fermented dairy products, demonstrated excellent milk fermentation properties when combined in laboratory experiments (Feng, 1986).In this study, we aimed to investigate the impact of different inoculum ratios of these 2 strains on fermentation time and post-acidification.In addition, we compared the viability of the LAB, acidification activity, volatile compounds, rheological properties, organic acid profiles, and post-acidification of yogurt fermented by their mono-cultures and cocultures.Our comprehensive analysis would provide valuable insights into the physiological and interactive properties of these bacteria strains and guide their application in large-scale industrial production of yogurt.

Preparation of Yogurt
Both S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047, stored at −80°C, were pre-cultured in sterilized milk at 36 ± 1°C for 15 h.The first precultures of each bacterium were then inoculated (3%; vol/vol) into sterilized milk and incubated at 42 ± 1°C for 8 h to obtain the second precultures as seed liquids.For yogurt production, the standard initial inoculum size for all samples was 5 × 10 6 cfu/g.Different proportions of the seed liquids were inoculated into sterilized milk using the ratios of CICC 6038:CICC 6047 as 1:1, 2:1, 10:1, 19:1, 50:1, and 100:1, and incubated at 42 ± 1°C.The specific inoculum size of CICC 6038 and CICC 6047 for 6 groups was provided in Table S1.The fermentation process was terminated when the pH value reached 4.6, and all fermented samples were stored at 4 ± 1°C. 150 mL of samples were collected at various predetermined time points during fermentation and refrigerated storage for subsequent analysis.In particular, yogurt samples fermented using the optimal proportions of a single strain (CICC 6038: St, or CICC 6047: Lb) and mixed strains (CICC 6038: CICC 6047 = 19:1, St+Lb) were prepared in the same way.The process of yogurt production and the parameters measured were illustrated in Figure S1.

pH Value and Titratable Acidity Determination
The pH value of the samples was continuously monitored during fermentation using a fermentation monitor (iCinac, French) until it reached 4.6, following the ISO 26323:2009(E) method.A pH meter (Mettler Toledo, USA) was used to measure pH value during storage.

Enumeration of Viable Cell
The viable cell counts of S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047 in the samples were determined using the plate pouring method, and the components of the medium used were shown in Table S2.Acidified MRS medium was used for the enumeration of Lb. bulgaricus by adjusting pH value to 5.4 to inhibit the growth of S. thermophilus (Kristina et al., 2019).For the enumeration of S. thermophilus, the M17-S medium was used, and glucose was replaced with sucrose to prevent the growth of Lb. bulgaricus (Silva et al., 2004).Sampling was done at various time intervals of FI, F2h, F4h, F6h, FT, S1d, S7d, S14d and S21d.

Organic Acids Analysis
High-performance liquid chromatography (HPLC) method was used to analyze the organic acid profiles of yogurt samples (Ashwell R Ndhlala et al., 2022).The samples were collected at various time points of FI, F2h, F4h, F6h, FT, S1d, S7d, S14d and S21d.For the analysis, a portion of 1.2 g of each sample was mixed with 8.8 mL of 0.01 mol/L sulfuric acid and then centrifuged at 10000 r/min for 10 min.Afterward, the supernatant was filtered using a 0.22 µm membrane.The organic acids were separated using Rezex ROA Organic Acid H + column (7.8 mm × 300 mm, 8 µm, Phenomenex, UK) at 80°C and subsequently detected using a photo-diode array detector (Ultimate; Thermo Fisher, USA) at a wavelength of 210 nm.The mobile phase was a 5 mM sulfuric acid aqueous solution, and 10 µL sample was extracted from the column at a flow rate of 0.6 mL/min.The peaks of organic acids were identified based on the retention time, and the content was determined using an external standard curve.The calibration curves were constructed using 5 concentration points.In addition, L-lactic acid/D-lactic acid was analyzed using a CRS10W column (300 mm × 4.6 mm × 3 µm).The extracted sample was injected (volume: 10 µL) in a CRS10W (300 mm × 4.6 mm × 3 µm), and 2 mmol/L CuSO 4 solution was used as mobile phase at a flow rate of 0.5 mL/min at 25°C.

Rheological Property Analysis
Rheolaser Master TM (Formulaction, France) was used to analyze the gel formation of milk during the fermentation process by multi-speckle diffusing wave spectroscopy (MS-DWS).At the start, 20 mL of inoculated milk samples were poured into glass tubes of inner diameter sizes of 27.5 mm, which were then placed in the Rheolaser Master apparatus previously equilibrated at 43°C.The milk was allowed to acidify until the pH value reached 4.6, then the monitoring was stopped, and the samples were stored overnight at 4°C before further measurements were taken.After allowing the samples to recover to room temperature (25°C), measurements were taken every 1 min using the Rheolaser Master.The software attached to the instrument was used to collect and analyze the original data to obtain various rheological parameters, including the macroscopic viscosity index (MVI), solid-liquid balance (SLB), elastic index (EI), and fluidity index (FI).

Analysis of Volatile Flavor Compounds by SPME-GC-MS
The relative content of each volatile flavor substance was determined using the peak area normalization method with computer retrieval.Standard mass spectra provided by Smart Aroma Database (https: / / www .shimadzu.com/an/ products/ gas -chromatograph -mass -spectrometry/ gc -ms -system/ smart -aroma -database/ index .html)was used for identification.To prepare the sample for this analysis, 5 mL of fermented milk and 2 mL of saturated sodium chloride solution were first sealed in a 20 mL glass vial.SPME: .The extraction head was aged at 250°C for 5 min, followed by an equilibrium temperature of 50°C for 45 min.Desorption was carried out for 3 min at 250°C.GC: .An SH-PolarWax column (60 m length, 0.25 mm inside diameter, 0.25 mm film thickness) was used.A programmed heating method was adopted, with an initial temperature of 40°C for 5 min, followed by an increase to 250°C at the rate of 3°C/min, and maintained for 15 min.The vaporization chamber was also maintained at a temperature of 250°C.The carrier gas used was helium at a flow rate of 1.0 mL/min, and split injection was performed.
MS: .The mass spectrometer (MSD) was operated in both full scan and MRM modes.Electron ionization was applied at a voltage of 70 eV, with a mass acquisition range (m/ z) from 35 to 400.The ion source temperature was maintained at 200°C, and the emission current was set to 100 µA.

Relative Odor Activity Value Calculation
The relative odor activity value (ROAV) was calculated according to a previously described method (Yi-

Electronic Nose Analysis
The electronic nose (e-nose) system used for this study was Fox 4000 e-nose system (ALPHA MOS, Toulouse, France) with 3 sensor matrix chambers and 18 metal oxide sensors.Due to the samples are edible foods, 2 sensor matrix chambers (Chamber A and Chamber B) and 12 metal oxide sensors (T30/1, P10/1, P10/2, P40/1, T70/2, PA/2, P30/1, P40/2, P30/2, T40/2, T40/1, and TA/2) were selected.All metal oxide sensors used and their main applications were listed in Table 2 (Min Xu et al., 2017).Each sample (5 g) was accurately weighed and sealed in 20 mL centrifugal bottles, incubated at 60°C, heated for 5 min, and then measured with the 12 metal oxide sensors.The environmental conditions for measuring the samples were set at (i) a sensor cleaning time of 120 s, (ii) an internal flow rate of 150 mL/min, and (iii) a sample detection time of 120 s.

Sensory Evaluation
To evaluate the sensory characteristics of the different fermented milk samples, 10 trained panelists used a 100-point intensity scale, modified based on the requirements specified in the Chinese dairy industry guideline RHB 103-2004(Zhe Zhang et al., 2021).The panelists scored acidity, sweetness, viscosity, and denseness on a scale of 0 -15 and taste, flavor, and texture separately on a 20-point intensity scale.Overall, the mean score for each sample was calculated by averaging the scores of all participants.

Statistical Analysis
All experiments were conducted in triplicate using independent cultures.Statistical analysis was performed using unpaired 2-tailed Student's t-tests.The results were represented as the mean value ± standard deviation (SD), and a p-value < 0.05 was considered statistically significant.Pearson correlation coefficient was used to calculate the correlation.Principal component analysis(PCA),heatmap visualization and variable importance in projection (VIP) scores by partial least squares-discriminant analysis (PLS-DA) were performed in MetaboAnalyst 5.0 (https: / / www .metaboanalyst.ca).The methodology for constructing a statistical model to evaluate the optimal ratio was provided in Supplemental File S1.

Effect of Various Inoculum Ratios on Acidification Activity and Post-acidification
The microbial ratio of yogurt starter cultures significantly affects the symbiotic strength between them, which in turn, determines the fermentation characteristics and post-acidification.To determine the optimal microbial ratio for efficient proto-cooperation and controlled post-acidification, 6 different inoculum ratios of CICC 6038 and CICC 6047, i.e., 1:1, 2:1, 10:1, 19:1, 50:1, and 100:1, were investigated.The results showed that all 6 ratios were able to successfully convert milk into curd through the fermentation process.However, the t pH4.6 and ∆pH during refrigerated storage differed based on the proportions.For instance, the t pH4.6 for 6 ratios ranged from 5.3 -7.1 h, and the higher the ratio of St to Lb, the longer the time to reach the terminal point (Figure 1A).After the 50 d of refrigerated storage, a higher ratio of St to Lb corresponded with a lower ∆pH.In particular, the pH values of yogurt fermented using the 10:1, 19:1, and 50:1 ratios were between 4.2 and 4.4 (Figure 1B), indicating the satisfactory organoleptic qualities (Hilde et al., 2003).

Determination of Optimal Ratio through Mathematical Modeling
The pH values obtained during yogurt fermentation process by different inoculum ratios of S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047 (1:1, 2:1, 10:1, 19:1, 50:1, 100:1) were used to generate primary fermentation characteristic models based on the modified Gompertz (Figure 1C) and modified logistic equations (Figure 1D).The corresponding model parameters were presented in Table S3.As expected, A tpH4.6 , µ m, tpH4.6 and λ tpH4.6 diminished with the increasing inoculum size of CICC 6038.The ∆pH data obtained during the storage of yogurt fermented by 6 inoculum ratios were utilized to generate post-acidification models based on the above-mentioned modified equations.The model parameters obtained were presented in Table S4.
The modified Gompertz model exhibited slightly higher R 2 values, suggesting it to be the most suitable primary model for all 6 inoculum ratios.The modified exponential function was chosen as a general secondary model for the A tpH4.6 , µ m, tpH4.6 and λ tpH4.6 parameters (Table S5).There were no significant deviations observed between the fitted curves and the observed values, indicating that the secondary models were suitable for predicting A tpH4.6 , µ m, tpH4.6 and λ tpH4.6 within the experimental inoculum ratio range.
After the parameter estimation and model discrimination, the functional relationship between t pH4.6 and inoculum ratio could be obtained from above 2 models as shown in Figure 1E.We could calculate Score value in the evaluation models with normalized t pH4.6 and ∆pH data (Figure 1F).Based on the evaluation model, the Score of 6 ratios were presented in Table 1.The 19:1 ratio exhibited the lowest Score among the 6 ratios studied, identifying it as the optimal ratio of CICC 6038 to CICC 6047.

Effect of Optimal Inoculum Ratio on Physicochemical Characteristics
The t pH4.6 for St and Lb mono-cultures were 9 and 7.2 h, respectively.In contrast, the time required for the cocultures of St+Lb was 6.6 h (Figure 2A), indicating that the fermentation speed of the cocultures was significantly faster.With an initial inoculation dose of 10 6 cfu/g, the viable bacterial count of all 3 groups reached 10 8 cfu/g by the end of the fermentation process (Figure 2B).The titratable acidities of St, Lb, and St+Lb groups were found to be 89.5°T,90.5°T, and 93.5°T at the end of fermentation (Figure 2C) and the 3 groups showed an average acid production rate (△°T/h) of 9.94, 12.4, and 14.2, respectively.
The St group exhibited exponential growth in the first 2 h, reaching maximal growth at 4 h.During this period, it produced acid more quickly than the other 2 groups.However, once the pH value dropped to 5.2 -5.5, the growth of S. thermophilus was suppressed, the pH value gradually fell, and acid production slowed.For the Lb group, growth was initially suppressed but later exhibited an exponential increase from 2 -4 h, after which the growth rate gradually declined.The pH value and TA exhibited gradual changes during the lag phase, followed by a substantial decrease in pH value and an increase in acidity, particularly during 4 -6h.For the St+Lb group, the acidification activity was in between St and Lb groups during the first 2 h, reached a maximum during 2 -4 h, and then the rate declined slightly until the end of the fermentation process.Throughout the entire storage period, post-acidification was lowest in St group, followed by the St+Lb group and then the Lb group (Figure 2D, Figure 2E).Notably, at the end of storage, the cocultures improved the survival activity of each LAB strain (Figure 2F).

Effect of Optimal Inoculum Ratio on Organic Acids Profiling
In this study, to investigate the impact of yogurt cultures on the organic acid profiles, the HPLC technique was used to determine the content and changes of L-/Dlactic acid, citric acid, formic acid, acetic acid, pyruvic acid, and succinic acid throughout fermentation and storage periods.Pearson correlation coefficient analysis was used to explore the correlations between organic acid metabolism and physicochemical properties.
We found that lactic acid was the dominant product of lactose catabolism by yogurt culture.Figure 3A showed that lactic acid increased remarkably as the fermentation period progressed for both mono-and coculture fermentation.The results of milk fermented by mono-culture revealed that S. thermophilus mainly synthesized L-lactic acid, and D-lactic acid was the main lactate product for Lb.bulgaricus.Fermentation with cocultures resulted in a combination of L (+) and D (−) lactic isomers, and it could be concluded that they were predominantly produced by S. thermophilus and Lb.Bulgaricus respectively.During fermentation, the mono-culture of S. thermophilus exhibited a stronger lactic acid-producing ability than Lb.bulgaricus in the early stage, while the mono-culture of Lb. bulgaricus initiated rapid acidification after the lag phase and generated the highest amount of lactic acid at the end of fermentation.Over the 21-d refrigerated storage, the content of L-lactic acid for the St group and St+Lb group decreased by 134 µg/g and 277 µg/g, respectively.In contrast, D-lactic acid increased by 760 µg/g in the Lb group and 348 µg/g in the St+Lb group (Figure 3B).Pyruvic acid was present in low concentrations in milk, and increased during the fermentation period for the 3 groups (Figure 3C).Notably, the amount of pyruvic acid in milk produced by mono-culture of Lb. bulgaricus was lowest during the refrigerated storage period (Figure 3D).Citric acid, a natural component of original milk, remained almost unchanged throughout the whole process in all samples (Figure 3E and Figure 3F).Succinic acid was detected in the samples fermented by yogurt starter culture containing Lb. bulgaricus, and the concentration in the Lb group was generally higher than in the St+Lb group (Figure 3G and Figure 3H).Acetic acid was detected in samples of Lb and St+Lb groups during the storage, and its content continuously increased until the end of the storage period (Figure 3I).Formic acid was not detected during the fermentation or storage period under the conditions used in this study.
Pearson correlation coefficients between the dynamics of each organic acid and the physicochemical properties of cell count and pH value were presented in Table S6.No significant correlation was observed between citric acid and viable bacteria count or pH value throughout the fermentation and storage process for all 3 groups.For the St group, L-lactic acid exhibited a significant negative correlation with pH value (P < 0.01) during the fermentation period.Whereas for the Lb group, both D-lactic acid and succinic acid showed significant positive correlations with the viable bacterial count (P < 0.01) and negative correlations with pH value (P < 0.01) during the fermentation period.Notably, during storage, acetic acid and pyruvate were significantly negatively correlated with pH value (P < 0.05).For the St+Lb group, D-lactic acid and L-lactic acid displayed significant correlations with the viable count of Lb. bulgaricus (P < 0.001) and S. thermophilus (P < 0.05), respectively, and L-lactic acid was significantly negatively correlated with pH value (P < 0.001).These results suggested that the organic acids produced by S. thermophilus played a more dominant role in the acidification process of yogurt during fermentation.In addition, there was a significant correlation between succinic acid and the viable count of Lb. bulgaricus (P < 0.05).However, during storage, only the content of acetic acid was found to be significantly negatively correlated with pH value (P < 0.05).

Effect of Optimal Inoculum Ratio on Rheological Properties
The MVI value indicated the microscopic viscosity characteristics of the samples.As shown in Figure 4A, the MVI values for the St+Lb group and St group remained low and fluctuated until 3.7 h.After that, the MVI values for both groups started to increase as the pH value dropped to the gel point, where the casein in milk began to form a gel structure.However, the MVI value of the Lb group raised 1.7 h later than the other 2 groups.Upon reaching the gel point, the pH value for both the St+Lb group and Lb group continued to drop rapidly, leading to a sharp rise in the MVI value of these samples, which peaked around 6.6 and 7.2 h, respectively, and ended the fermentation process.In contrast, the pH value for the St group dropped slowly after reaching the gel point and ended the fermentation process after 9 h.At the end of the fermentation process, the MVI value for the St+Lb group was intermediate between the St and Lb groups.After postripening, the MVI value for the St group decreased while the MVI values for both the St+Lb group and Lb group increased.However, the St group still exhibited a higher MVI value than the other 2 groups.
The consistency between EI and MVI values was observed (Figure 4B).The samples exhibited stable EI values until reaching the gel point, after which they rapidly increased and reached their peak.Notably, the EI value increased for the St+Lb group and St group, but decreased for the Lb group after post-ripening.
The texture of the fermented milk was elastic and solid-like when SLB was between 0 and 0.5, whereas it was viscous and liquid-like when SLB was between 0.5 and 1.The SLB values of 3 groups fluctuated before reaching the gel point, but stabilized between 0.4 and 0.45 and remained constant until the end of fermentation.After post-ripening, the SLB value for St+Lb group and St group remained below 0.5, suggesting that the samples maintained a stable solid state (Figure 4C).The FI value for all groups showed significant fluctuations at a high level before the milk reached the gel point, signifying that the sample was still in the liquid state (Figure 4D).After that, the FI value for all 3 groups declined rapidly and stabilized at a low level until post-ripening.These observations were consistent with the SLB values observed for each group.
In this study, PCA, an unsupervised multivariate statistical analysis method, was utilized to disclose substantial differences in the metabolites among the 3 groups, as illustrated in Figure 6A.Principal components P1 and P2 accounted for 82.5% and 12.1% of the variations, respectively.The heatmap outcome demonstrated a clear differentiation among the 3 groups, as shown in Figure 6B.

Effect of Optimal Inoculum Ratio on Sensory Characteristics
The results of PCA derived from e-nose, a valuable tool for rapidly evaluating volatile flavor compounds in fermented dairy products, demonstrated that PC1 accounts for 99.3% of the variation among experimental samples, while PC2 accounts for 0.5%, collectively explaining 99.8% of the total variance (Figure 6C).The PCA chart clearly revealed significant differences in flavor profiles across the 3 groups.Additionally, the heightened sensor responses (P10/1, P30/2, P30/1, T30/1, P40/2, and PA/2) indicated a substantial presence of organic and alcohol compounds in the samples (Figure 6D).

Effect of Optimal Inoculum Ratio on Sensory Evaluation
As shown in Figure 6E, the sample fermented using coculture achieved a higher overall score in comparison to those fermented with mono-culture.Even though the sensory profile of S. thermophilus mono-fermented milk was similar to the coculture sample, the latter produced a more desirable texture and viscosity.Therefore, we confirmed that coculture fermentation presented certain advantages over mono-culture fermentation.

DISCUSSION
Yogurt is a microecosystem which relies on the mutualistic interaction between 2 defined cultures of S. thermophilus and Lb.bulgaricus for its successful manufacture (Pascal Courtin and Françoise Rul, 2004).The positive interaction depends on the specific strains used, making it crucial to select suitable strain combinations.Additionally, the relative inoculum size of yogurt bacteria had significant impact on the strength of their symbiotic relationship, leading to variations in fermentation properties of yogurt.Therefore, it is of great importance to determine the optimal inoculum ratio for each bacteria consortium to achieve specific yogurt properties.However, the influence of different inoculum ratios on yogurt properties has been rarely reported.In this study, we focused mainly on the fer-mentation time and post-acidification level as evaluation indicators.Though constructing the statistical model for selecting the optimal ratio, which has not been previously reported, we determined that the ratio of 19:1 as the appropriate initial proportion of S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047 in milk for demonstrating our satisfactory outcomes.
In this study, we performed the comparative analysis of fermentation characteristics and storage stability of milk fermented by their mono-cultures and coculture at 19:1.It was observed that both S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047 strains were capable of individually fermenting milk to form curd.However, when combined at optimal ratio, they exhibited a synergistic effect that reduced the fermentation time, generated the highest biomass, and increased the acid  production rate in yogurt compared with using either strain alone.The proto-cooperation is partly due to S. thermophilus CICC 6038's ability to quickly metabolize lactose and produce acids.However, it was sensitive to acidity and experienced significant growth restriction after around pH 5.5, as reported in previous study (C.Gasser et al., 2022, Kristina et al., 2019).On the other hand, Lb. bulgaricus was better adapted to the acidic environment and dominated the fermentation process subsequently until the endpoint.Additionally, we observed the enhanced fitness of S. thermophilus during storage and a higher survival activity of Lb. bulgaricus at the end of storage when cocultured with S. thermophilus.This improvement could potentially be attributed to the increased synthesis of EPS during coculture fermentation.EPS acts as a bio-stabilizer in yogurt, making the strain more resilient to harsh conditions (Robina Taj et al., 2022;Swati Tiwari et al., 2021), and it also contributes to the texture, firmness, and viscosity of the final product (Nguyen et al., 2019;Cui et al., 2017;Ren et al., 2016).We also confirmed that Lb. bulgaricus was responsible for the level of postacidification in the yogurt as it continued to slowly acidify under low-temperature conditions.Studies have shown that various strains exhibit significant differences in the number and expression of genes responsible for post-acidification capacity, including key enzymes in glycometabolism and proteolysis (Yingxue Yue et al., 2022).Currently, the most promising and economical way to control post-acidification is to screen aciditysensitive Lb. bulgaricus strains at room temperature.Organic acids contribute to the characteristic sensory properties of fermented dairy products and act as natural preservatives.Three fermentation models  showed different organic acid metabolism progressions.
To our knowledge, this study was the first to use the HPLC method to systematically compare the dynamic changes of D-lactic acid and L-lactic acid in mono-and coculture fermentation processes.The formation of the 2 isomers of lactic acids depends upon the fermentation strains, milk composition, temperature, pH value, storage temperature, storage time, etc (Livia Alm, 1982).S. thermophilus CICC 6038 specifically produced L-lactic acid with good sensory properties and stability, showing its great potential for use in infant yogurt production.Notably, we found a significant positive correlation between L-lactic acid content and viable cell counts of S. thermophilus during coculture fermentation.Similarly, there was a significant positive correlation between D-lactic acid content and viable cell counts of Lb. bulgaricus.These findings suggest that monitoring the production of L-lactic acid and D-lactic acid could serve as an indicator to evaluate strain efficiency, demonstrate the dynamic relationship of starter culture and understand the fermentation process of yogurt.The proportion of L-lactic acid ranged from 80 -73% during storage, exceeding the 2:1 ratio of L-lactic acid to D-lactic acid in commonly defined quality yogurt (Tamime, A.Y. et al., 1999).The coculture fermentation achieved lower content of D-lactic acid in the final product.Succinic acid is a component of the tricarboxylic acid (TCA) cycle and can contribute to the typical aroma of fermented dairy products.It was the differentiated metabolic product to discriminate St group from Lb and St+Lb in this study, probably because Lb. bulgaricus possessed redundant genes coding the fumarate reductase enzyme, which could metabolize fumaric acid to succinic acid, while this gene is rarely observed in the genome of S. thermophilus (Pascal Hols et al., 2005).A recent study (Eri Yamamoto et al., 2021) demonstrated that S. thermophilus commonly produce fumaric acid, which can be utilized by Lb. bulgaricus during coculture.The production of acetic acid during yogurt fermentation is limited by the low pyruvate levels and the high metabolic demand for lactate production.However, our results showed that the Lb group and St+Lb group exhibited an accumulation of acetic acid during refrigerated storage, which had a stronger correlation with post-acidification than lactic acid.This discovery reveals new insight into the mechanism behind post-acidification and provides possible guidance to inhibit it by targeting the enzymes involved in the acetic acid synthesis.
The profiles of volatile flavor substances play a crucial role in determining the taste and aroma of fermented milk products.The production of these metabolites varies not only among different species but also among strains within the same species (Gezginc et al., 2015).In this study, we used SPME-GC-MS analysis to investigate the metabolic ability of S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047.Our results confirmed that the coexistence of these 2 bacteria in milk led to a synergistic alteration of aroma compounds in the final product.Our findings are consistent with pre-  vious studies, which have reported that the symbiotic relationship between Lb. bulgaricus and S. thermophilus can boost the content of acetaldehyde and 2,3-butanedione (Yaru et al., 2021).Acetaldehyde is a key volatile compound in fermented milk with a green apple flavor, formed by the decarboxylation of pyruvate.The content of acetaldehyde in the coculture fermented samples was significantly higher, resulting in a ROAV value of 18.28, compared with 3.8 and 5.86 in the mono-culture fermented samples.2,3-Butanedione, produced by 2-acetolactate, can provide the yogurt a sweet, buttery and milky aroma (Huaixiang et al., 2020).Our findings also demonstrated the same synergistic effect in the production of decanal, providing a floral flavor (Chen et al., 2022), with a ROAV value of ≥ 1 in coculture fermented sample but < 1 in the other 2 sample groups.
3-hydroxy-2-butanon, also known as acetoin, is mainly converted from 2,3-butanedione, which imparts sweet, cultured, and buttery aromas of yogurt (Zhe Zhang et al., 2021).It was not detected in Lb group, perhaps diacetyl reductase or acetolactate decarboxylase was absent in some Lb.bulgaricus strains (Beshkova et al., 2003).Besides, esters, produced through the esterification of fatty acids and alcohols, play a crucial role in shaping the fruity properties of dairy products, particularly ethyl ester (P.M.G.Curioni and J.O. Bosset, 2002).Our analysis showed that ethyl acetate, with a ROAV value of 1.27, has a unique and significant volatile flavor component in the coculture fermented sample with a pineapple aroma.The metabolic pathways of part key volatile metabolites and organic acids were summarized in Figure 7.
Texture is a crucial factor that impacts the quality of yogurt.Rheological analysis, performed under zero shear conditions, is an effective method to evaluate yogurt texture changes while preserving its delicate structure (Carlotta Ceniti et al., 2019).This modern passive microrheology technique has been recently implemented to assess the effects of probiotic strains (Mei et al., 2020) and resistant starches (Jun He et al., 2019) on the yogurt properties.In this study, we first utilized the modern rheological method to monitor the monoculture and coculture fermentation process, enabling us to observe real-time differences in the gelation among the 3 fermentation models.The EI value provides a direct measure of the elastic modulus as it changes over time, with its stability indicating the stability of fermented milk samples.During the initial stages of fermentation, the milk particles move rapidly until they form a solid-like substance, representing the gel point (Carlotta Ceniti et al., 2019).Compared with Lb group, the MVI and EI values for the St group increased rapidly in the early fermentation stage and reached the gel point faster.However, as fermentation progressed, the acid production rate for Lb group accelerated, resulting in faster attainment of the fermentation endpoint and gel formation.Coculture fermentation combined the benefits of faster gel start time and rate, leading to quicker attainment of the fermentation endpoint, as evidenced by pH value changes in all 3 fermentation groups.By the end of the fermentation process, the EI value for St+Lb group was significantly higher than that of the other 2 samples, revealing that coculture fermentation with S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047 led to significantly greater yogurt stability than mono-culture fermentation.The SLB value indicates the ratio of solid and liquid textures in a sample, with a lower slope indicating slower milk particle movement.The FI value reflects the flow speed of particles in fermented milk, with SLB and FI values directly proportional to particle movement and sample fluidity (Jun He et al., 2019).In this study, we have shown that yogurt produced through coculture fermentation demonstrated higher elastic and lower moduli than mono-culture groups.Coculture fermentation improved the gel structure strength and increased the yogurt's elasticity and solid-like properties.

CONCLUSIONS
In this study, we highlighted the synergistic effects of specific strains of S. thermophilus and Lb.bulgaricus on yogurt properties and emphasized the importance of selecting appropriate proportions.By developing a mathematical model to evaluate 6 experimental ratios, we determined that the ratio of 19:1 (S. thermophilus CICC 6038 and Lb.bulgaricus CICC 6047) is optimal for achieving favorable fermentation performance and enhanced post-acidification.Compared with monocultures, the coculture demonstrated higher acidification activity, improved rheological properties, richer volatile compounds, more desirable sensorial qualities, and higher survivability of Lb. bulgaricus at the end of storage.We believe that the findings of this study have implications for optimizing the production of yogurt and other fermented foods, as well as enhancing our understanding of microbial symbiosis for broader applications in diverse bioprocesses.
Figure 1.Dynamic changes of pH during yogurt fermentation (A) and refrigerated storage (B), by different proportions of CICC 6038 and CICC 6047 cocultures for Yogurt fermentation, including 1:1, 2:1, 10:1, 19:1, 50:1, 100:1.Observed pH profiles (symbols) during fermentation process fitted with the primary models (lines) modified Gompertz equation (C) and modified logistic equation (D).Predicted with different inoculum ratios of S. thermophilus CICC 6038 to Lb. bulgaricus CICC 6047 based on parameters of primary models and secondary models for yogurt fermentation characteristics (E).Evaluation models lines (F), including (a) curve of ∆pH and inoculum ratio (b) curve of Score and inoculum ratio and (c) curve of t pH4.6 and inoculum ratio.

Figure 5 .
Figure 5. Types of volatile flavor compounds in the 3 groups of samples (A); The amount of volatile flavor substance was different among the 3 groups (B)

Figure 6 .
Figure 6.Differences in volatile metabolome and sensory evaluation between mono-culture and coculture fermented milk.(A) Principal component analysis (PCA) score plot showing volatile metabolomes of 3 groups; (B) Heatmap of identified volatile metabolites in each group.PCA (C) and VIP (D) scores by PLS-DA of E-nose data; (E)Radar fingerprint chart of sensory evaluation of 3 groups.

Table 3 (
Ge et al.: Fermentation characteristics and… Continued).Types and relative contents of volatile flavor compounds Ge et al.: Fermentation characteristics and…