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Article

Effects of Lactobacillus helveticus H11 Addition on the Fermentation, Storage, and Probiotic Properties of Brown Fermented Milk

by
Wenkang Zhai
1,2,3,
Chunle Tian
1,2,3,
Huimin Chen
1,2,3 and
Jianli Li
1,2,3,*
1
Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot 010018, China
2
Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Hohhot 010018, China
3
Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot 010018, China
*
Author to whom correspondence should be addressed.
Foods 2026, 15(4), 606; https://doi.org/10.3390/foods15040606
Submission received: 4 January 2026 / Revised: 2 February 2026 / Accepted: 5 February 2026 / Published: 7 February 2026
(This article belongs to the Section Food Microbiology)
Browse Figures
Versions Notes

Abstract

This study investigated the effects of adding Lactobacillus helveticus H11 on the fermentation characteristics, storage performance, and probiotic functionality of brown fermented milk. The results were obtained by analyzing the microrheological properties during the fermentation process, as well as changes in titratable acidity, viable cell count, texture, ACE (angiotensin-converting enzyme) inhibition rate, and metabolite profiles during storage. The findings indicated that the addition of H11 significantly shortened the fermentation time of brown fermented milk and improved its gel structure. Simultaneously, it delayed the post-acidification phenomenon during storage, enhancing storage stability. Moreover, it enhanced the ACE inhibitory activity of the product, demonstrating potential antihypertensive health benefits. Metabolomic analysis further revealed that H11 promoted the production of flavor compounds, such as phenylacetaldehyde and phenyllactic acid, enriching the flavor diversity. Additionally, it concurrently regulated the metabolism of tyrosine and vitamin B6. These discoveries provide a scientific basis for the commercial development of brown probiotic products.

1. Introduction

The integration of probiotics into dairy products has garnered increasing interest due to their potential to enhance the sensory attributes, nutritional profile, and physical-chemical properties of these food items. Probiotics have been effectively utilized to streamline production processes, elevate product quality, and confer health benefits.
For instance, the addition of specific probiotic strains, such as Bifidobacterium animalis subsp. lactis Probio-M8, has been demonstrated to reduce fermentation times significantly, thereby offering economic advantages in industrial settings [1]. Similarly, the utilization of Lactobacillus plantarum A3 in yogurt production has been linked to improvements in texture characteristics, including firmness, consistency, and viscosity [2]. Beyond enhancing texture and accelerating fermentation, probiotics also play a crucial role in modulating acidity levels during the fermentation process and maintaining activity under low-temperature storage conditions, which mitigates excessive acidity buildup and pH fluctuations [3].
Furthermore, probiotics contribute to the nutritional value and health benefits of fermented milk through their metabolic activities. They produce beneficial metabolites like propionic acid and vitamins, and active enzymes that break down proteins into bioactive peptides with antioxidant, anti-inflammatory, and hypotensive properties [4]. These metabolic changes not only enrich the nutritional content of dairy products but also sustain their functional activity.
A particularly noteworthy probiotic strain, Lactobacillus helveticus H11 (H11), was initially isolated from Xinjiang sour mare milk. Preliminary studies have indicated that H11 not only exhibits robust fermentation capabilities but also enhances the storage stability of fermented milk products. Moreover, H11 significantly boosts the production of bioactive peptides, namely Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which possess angiotensin-converting enzyme (ACE) inhibitory activity [5]. This activity is crucial for lowering blood pressure and managing hypertension, making H11 a valuable asset in the development of functional foods and health products [6,7].
The primary ingredients used in the preparation of brown fermented milk include reducing sugars and raw cow milk (or reconstituted skimmed milk powder). Upon thermal treatment, high-temperature browning occurs via the Maillard reaction; this is subsequently followed by lactic acid fermentation driven by selected strains of lactic acid bacteria, resulting in a distinctive flavored fermented milk product [8]. This type of fermented milk is abundant in a variety of nutrients, including essential vitamins, minerals, and high-quality proteins, as well as a broad profile of indispensable amino acids, which are more readily absorbed and utilized by the human body. But brown fermented milk often struggles with issues related to acidity, texture, and flavor, which detract from its consumer appeal and market competitiveness. Sensory quality is a key determinant of product acceptance, and evidence suggests that probiotics can enhance flavor by generating a range of pleasant flavor compounds during fermentation and storage [9,10]. For example, the co-use of Streptococcus thermophilus and Lactobacillus helveticus in fermented milk production has been shown to yield flavor compounds such as benzaldehyde, 2-undecanone, and linalool, which contribute to a more desirable sensory experience [11,12]. These findings suggest that the addition of H11 may enhance the flavor of brown fermented milk, meeting the dual consumer demands for taste and nutrition.
In this study, we employed PYS-010, composed of Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus as the base starter culture and subsequently added H11 to prepare brown fermented milk. We conducted a comprehensive analysis of various parameters, including pH, titratable acidity (TA), viable cell count, ACE inhibition rate, and metabolomic profile, to compare brown fermented milk samples produced with and without H11. Our objective was to investigate the alterations induced by H11 during fermentation and storage and to gain insights into the underlying mechanisms. A thorough understanding of these effects could facilitate the utilization of H11 as a functional ingredient in brown fermented milk and support its commercial development as a health-promoting product.

2. Materials and Methods

2.1. Experimental Strain

H11 was obtained from the Lactic Acid Bacteria Cell Collection at the Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering of Inner Mongolia Agricultural University, China. The commercial basic starter culture PYS-010—composed of S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus—was provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd. (Beijing, China).

2.2. Chemicals and Reagents

The following information of all reagents and materials used in the experiment is clearly provided, including sources and manufacturers: ACE (0.1 U/g) was purchased from Sigma (St. Louis, MO, USA); trifluoroacetic acid (TFA), hippuric acid (HA), hippuryl-histidyl-leucine (HHL, 25 mg), boric acid, and borax were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China); acetonitrile and methanol were purchased from Fisher Chemicals (Fair Lawn, NJ, USA); ultrapure water was purchased from Watsons Group Co., Ltd. (Hong Kong, China); pure milk was purchased from Inner Mongolia Mengniu Dairy (Group) Co., Ltd. (Hohhot, China); white sugar and glucose were purchased from Zhongliang Sugar Co., Ltd. (Zhongshan, China); and MRS agar medium was purchased from Haibo Biological Co., Ltd. (Qingdao, China).

2.3. Production of Brown Fermented Milk

Glucose was added to milk at a 4% (wt/wt) ratio, and the milk was browned at 95 °C for 3 h to obtain the base dairy component. After this component was cooled to 50 °C, 6.5% (wt/wt) white sugar was added, and the mixture was stirred evenly to ensure complete dissolution. Next, the mixture was homogenized at 17 MPa and pasteurized for 5 min after homogenization. The mixture was cooled to 42 °C, inoculated with PYS-010 and H11, and fermentation was considered complete when the pH reached 4.6. The product obtained at this stage was brown fermented milk. The brown fermented milk samples were stored at 4 °C for 21 days. The C group was inoculated with PYS-010 (0.003% wt/wt) and H11 (5 × 106 CFU/mL), while the S group was inoculated with 0.003% (wt/wt) PYS-010 only.

2.4. Microrheological Monitoring During Fermentation

First, 20 mL of the inoculated samples were placed in a specialized microrheology sample bottle (inner diameter: 27.5 mm; Rheplaser Master, Beijing, China) and added to the sample tank. The program was switched to the expert mode, and the temperature was set to 42 °C. During fermentation at 42 °C, parameters such as the elasticity index (EI), macroscopic viscosity index (MVI), solid–liquid balance (SLB), fluidity index (FI), and gel changes were monitored. Data were collected every 5 min until fermentation was complete and analyzed using the instrument’s in-house analysis software. Each sample was examined in triplicate [13].

2.5. Acidity Determination

The determination of pH was conducted as follows: the brown fermented milk samples were first retrieved from a 4 °C refrigerated environment and allowed to equilibrate to room temperature prior to measurement. The pH values were then precisely measured using a Leici Model SJ-3F pH meter (Mettler-Toledo GmbH, Greifensee, Switzerland). To ensure data accuracy, three parallel replicates were prepared for each sample.
For the determination of titratable acidity (TA), the method was based on that specified in previous studies, namely the phenolphthalein indicator method, to ensure the accuracy and reliability of the results [14]. Specifically, 5 g of brown fermented milk sample was accurately weighed into an Erlenmeyer flask, followed by the addition of 40 mL of freshly boiled and cooled distilled water for dilution. Subsequently, three drops of phenolphthalein indicator were added and the mixture was thoroughly homogenized. The diluted sample was titrated with 0.1 M NaOH solution while continuously swirling, until a faint pink color persisted for at least 30 s, indicating the titration endpoint. The TA value was calculated by multiplying the volume of NaOH solution consumed by a factor of 20. Each sample was analyzed in triplicate.

2.6. Texture Analysis

To comprehensively monitor the changes in the textural properties of brown fermented milk during storage, such as hardness, consistency, cohesiveness, and viscosity index, a TA.XT plus texture analyzer (Stable Micro Systems, Surrey, UK) was employed for detection. The specific operational procedure is as follows: First, pour the brown fermented milk sample into a dedicated TA.XT plus texture analyzer sample cup, ensuring that the pouring volume accounts for 70% of the sample cup’s capacity. Next, select the A/BE probe pressure plate (with a diameter of 35 mm) for precise detection. The program on the texture analyzer should be set to the yogurt consistency measurement method, wherein the pre-test speed, test speed, and post-test speed are 1.5 mm/s, 1.0 mm/s, and 1.5 mm/s, respectively. The initial trigger force is 2.0 g, the test distance is 20.0 mm, the compression stroke is 20%, and the compression duration is 5.0 s. Furthermore, to ensure the accuracy of the test results, the probe should be thoroughly cleaned before and after each detection to prevent interference from sample residues. Each sample should have three replicates.

2.7. Determination of ACE Inhibitory Activity

The brown fermented milk samples were centrifuged (8000× g, 10 min), and the supernatants were collected. The pH of all samples was adjusted to 8.3 using a 1 M NaOH solution, followed by a second centrifugation (8000× g, 10 min) to collect the supernatants for further use. Subsequently, 80 μL of the sample supernatant was added to a 1.5 mL EP tube containing the substrate hippuryl-histidyl-leucine (HHL) at a concentration of 5 mM, with 200 μL of HHL added. The mixture was pre-incubated at 37 °C for 10 min, and a blank group was established by replacing the sample solution with borate buffer. After that, an ACE solution (20 μL, 0.1 U/mL) was added, and the mixture was vortexed and incubated at 37 °C for 60 min. Finally, 150 μL of 1 M HCl was added to terminate the enzymatic reaction. The resulting mixture was then filtered through a 0.22 μm microporous membrane before being analyzed. Detection was performed using HPLC, with the maximum absorption peak of hippuric acid (HA) monitored at a wavelength of 228 nm.
The hippuric acid content in the enzymatic hydrolysis reaction products was determined using the RP-HPLC method to ensure accurate results. A ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm), manufactured by Agilent Technologies in the United States (Agilent, Santa Clara, CA, USA), was employed. A single mobile phase consisting of 22% (v/v) acetonitrile aqueous solution containing 0.1% trifluoroacetic acid was used. The flow rate was set at 1.0 mL/min, the column temperature was maintained at 30 °C, the detection wavelength was 228 nm, and the injection volume was 20 μL [15].

2.8. Determination of VPP and IPP Concentration

The brown fermented milk samples were subjected to centrifugation at 4500× g for 10 min, after which the supernatants were collected. The pH of all samples was uniformly adjusted to 8.3 using a 1 M NaOH solution, followed by a second centrifugation at 12,000× g for 10 min. The resulting supernatants were then filtered through a 0.22 μm microporous membrane prior to instrumental analysis.
The HPLC conditions were as follows: A C18 column (CAPCELL PAK MG, manufactured by Shiseido, Minato City, Japan) with dimensions of 2.0 × 100 mm and a particle size of 5 μm was employed. The flow rate was set at 0.2 mL/min, with an injection volume of 10 μL per run and a column temperature maintained at 30 °C. The detection wavelength was established at 210 nm. The mobile phase consisted of two components: Mobile phase A was deionized water containing 0.1% trifluoroacetic acid, while mobile phase B was pure acetonitrile also containing 0.1% trifluoroacetic acid.
For the ESI-MS/MS analysis, the mass spectrometer was operated in positive ion mode. The sheath gas flow rate was set at 35 arbitrary units (arb), and the auxiliary/purge gas flow rate was adjusted to 5 arb. The ion spray voltage was configured to 5.50 kV, with a capillary temperature of 300 °C and a capillary voltage of 16 kV. The collision energy for the second-stage collision was set at 30%, and the fragment scan range was defined between 100 and 400 m/z [16].

2.9. Non-Targeted Metabolomics

2.9.1. Metabolite Extraction

The brown fermented milk samples were removed from the −80 °C refrigerator and allowed to thaw at 4 °C. Subsequently, 3 mL of each sample was mixed with 1 mL acetonitrile to induce protein precipitation, and the samples were subjected to high-speed centrifugation (10,000× g, 10 min). Then, 1 mL of the supernatant was transferred to a new 5 mL EP tube, mixed with 3 mL of pure acetonitrile, and mixed thoroughly before incubation at 4 °C for 2 h. This was followed by a second round of high-speed centrifugation (12,000× g, 5 min). After centrifugation, the entire supernatant was removed and placed in a 2 mL EP tube; it was then concentrated and dried for 9 h using a vacuum centrifugal concentrator to ensure that the sample was fully processed. A 40% acetonitrile aqueous solution (400 μL) was quickly added to re-dissolve the concentrate. After thorough mixing, the sample was filtered using a 0.22-µm filter membrane and transferred to a sample injection vial with an inner liner. Three parallel replicates were prepared for each sample. Simultaneously, quality control (QC) samples were injected after every 10 testing for quality monitoring. Before testing each experimental sample, the QC sample was injected into the chromatographic system to ensure the system’s stability and reliability.

2.9.2. Test Conditions

The samples were analyzed using the UPLC-Q-TOF system (SCIEX 6600, Framingham, MA, USA). The samples were separated using an Acquity UPLC® HSS T3 column (Waters, Milford, MA, USA, 1.8 µm × 2.1 mm × 100 mm), with the column temperature set to 40 °C, flow rate set to 0.30 mL/min, and injection volume set to 2.0 μL. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B was an acetonitrile (0.1% formic acid) solution. The elution parameters were as follows: 0–1.0 min, 5% B; 1.0–8.0 min, 40% B; 8.0–15.0 min, 85% B; 15.0–18.5 min, 90% B; 18.5–20.5 min, 90% B, 20.5–22.0 min, 5% B; 22.0–23.0 min, 5% B. The ionization voltage was 5500 V, with gas 1 and gas 2 set to 50 and 35 psi, respectively, and the ion source vaporization temperature set to 550 °C.

2.9.3. Metabolomics Data Processing

Raw data were acquired in the continuous mode and processed using MasterView v1.21 For D7 (SCIEX, USA) and Progenesis QI software 2.1 (Waters, Milford, CT, USA). Data preprocessing included the peak alignment, peak recognition, and deconvolution steps.

2.10. Multivariate Statistical Analysis

Each group of samples was tested in at least three parallels. An independent samples t-test and ANOVA were to compare differences among the groups, with the significance level set to 0.05. Analysis and plotting were performed using Origin 2021 and R 4.3.2 software.

3. Results

3.1. Microrheological Monitoring of Brown Fermented Milk During Fermentation

During the fermentation process, brown fermented milk gradually transformed from a liquid to a semi-solid, and its internal structure changed significantly. As the probiotic strains continued to grow, the acidity of the fermented milk system decreased, the particle structure changed, and the casein micelles gradually transitioned toward a gel state. Therefore, the brown fermented milk samples in both the S and C groups exhibited significant changes in rheological properties. The gelation process of brown fermented milk samples was monitored using Diffusing Wave Spectroscopy technology (DWS) based on elasticity index (EI), macroscopic viscosity index (MVI), solid–liquid balance (SLB), and fluidity index (FI) values.
In this study, both groups of samples were in a liquid state at the 0–4 h stage, showing low elasticity (Figure 1A). During this phase, casein existed as a casein–calcium phosphate complex and did not form a gel network structure. As fermentation progressed, the pH value decreased gradually, the overall charge density of the colloidal system changed, and both the casein micelles and calcium phosphate complex transitioned to form a gel structure [17], reaching the gel point.
EI values are proportional to sample stability. Moreover, good stability helps reduce physical and chemical changes, such as whey separation, protein precipitation, and flavor changes, during product storage. The EI of brown fermented milk increased rapidly after gelation, reaching a high-elasticity stage, and the gel structure gradually became more stable. H11 addition increased the EI value of brown fermented milk and improved its stability.
Figure 1B illustrates the changes in the MVI of the two groups of samples during the fermentation process. The larger the MVI value, the longer it takes for particles to move within the system. Thus, higher MVI values indicate a higher system viscosity, which promotes the formation of a stable gel structure and improves storage stability [18].
The SLB refers to the ratio between the liquid and solid characteristics of fermented milk. An SLB of 0, 0–0.5, 0.5–1, and 1, respectively, indicates that the fermented milk is in a solid, solid or elastic, liquid or viscous, and liquid state. In this study, during the early stage of fermentation, the viscosity of the sample was low, and the sample was in a liquid state. As the acid production by the strains continued to increase, the SLB value changed significantly around 4 h. During this process, casein dissociated and gradually formed a gel structure. Subsequently, the SLB value fluctuated around 0.5, but the SLB value of group C remained lower than that of group S. At this point, casein aggregated and transformed into a colloidal state, reducing the solubility of the sample and increasing its viscosity. This was consistent with the changes observed in the EI and MVI values. The results showed that H11 addition helps increase the viscoelasticity of brown fermented milk and promotes its stability. Subsequently, the brown fermented milk gradually shifts toward a semi-solid state, leading to a more delicate and richer taste and texture.
The larger is the FI, the stronger is the fluidity of the sample, corresponding to a higher movement rate of particles in the system. A high flow factor (about 10 Hz) indicates that a sample has liquid-liked properties, while a low flow factor (about 10−2 Hz) indicates a solid state [19]. As shown in Figure 1D, after fermentation, the FI of the two groups of brown fermented milk remained unchanged, approaching 10−3~10−2 Hz. This indicated that the samples had solid-like characteristics at this point, and that the structure of the brown fermented milk had become stable and mature.

3.2. pH, TA, and Viable Cell Counts

TA and pH are two key indicators for evaluating the quality of fermented milk and are closely related to its texture and flavor. Severe post-acidification can cause a sharp decline in the quality and taste of fermented milk. In this study, the brown fermented milk samples continued to produce acid even during storage at 4 °C, causing the pH to exhibit a downward trend. At the beginning of storage, the pH values in groups S and C were 4.48 ± 0.000 and 4.51 ± 0.015, respectively. The changes in pH value were more noticeable at 1–7 days of storage in group C and at 14–21 days of storage in group S. After 21 days of storage (Figure 2A), the pH value of group C was significantly higher than that of group S (p < 0.05). Researchers have previously found that pH values between 4.0 and 4.6 during storage indicate that no significant post-acidification has occurred. This pH range is widely appreciated by consumers and is considered optimal for beverages [20]. The pH values of both groups of samples were within this optimal range even at the end of the storage period.
As storage was prolonged, both groups of brown fermented milk showed consistent alterations, with TA levels exhibiting a continuous upward trajectory. However, the TA of group S remained significantly higher than that of the C group throughout the storage period (p < 0.05) (Figure 2B). After 21 days of storage, the TA of the S and C groups were 99.6 ± 0.75 °T and 97.5 ± 0.10 °T, respectively. Donkor et al. [21] previously suggested that the optimal TA range of fermented milk is 70–110 °T, and consumers prefer products with a TA within this range. When the TA exceeds 120 °T, noticeable deterioration in taste and flavor may be observed in fermented milk. The TA of both the S and C groups remained below 100 °T during storage in this study. These findings also indicated that H11 has relatively stable acid-generating properties, does not negatively impact product quality during storage, demonstrates excellent fermentation potential, and can be used for the production of brown fermented milk products.
The viable cell count of brown fermented milk is a key consideration for the selection of probiotic starter cultures and can accurately reflect the survival of strains in the fermented samples. As shown in Figure 2B, the viable cell count of the samples showed an initial increase, peaking on the 7th day before decreasing gradually thereafter. After 21 days of storage, the viable bacterial counts in groups S and C were 8.0 × 108 and 7.3 × 108 CFU/mL respectively. Surprisingly, the viable bacterial counts in group S were significantly higher than those in group C throughout the storage period. This was consistent with the trend of TA (Figure 2B). These findings demonstrated that contrary to our expectations, H11 addition did not increase the viable cell counts of brown fermented milk. The reason underlying this phenomenon warrants further attention. Nevertheless, the viable cell counts in both groups of samples were greater than 108 CFU/mL, which is much higher than the threshold of 106 CFU/mL specified for the positive health benefits of probiotic foods [22].

3.3. Textural Changes in Brown Fermented Milk During Storage Time

Texture is an important physical property of brown fermented milk and directly affects taste, consumers’ experience, and market acceptance. The texture analysis of brown fermented milk can provide deeper insights into its stability during storage. Texture analysis typically includes four key indicators: cohesiveness, firmness, index of viscosity, and consistency.
Cohesion represents the density of fermented milk samples, describing its fluidity and reflecting the internal friction within the milk. As shown in Figure 3A, the cohesiveness of group C samples was higher than that of group S samples at all time points except day 1. The difference was significant on days 7 and 14 (p < 0.05), indicating that H11 addition can reduce problems such as whey precipitation.
Firmness reflects the degree to which brown fermented milk can recover its original shape after being exposed to an external force. When the sample has a dense protein network and gel-like firmness, its quality becomes more stable [23]. In group S, the firmness of brown fermented milk showed a downward time-dependent trend. Meanwhile, in group C, the firmness increased initially before gradually stabilizing. Thus, H11 addition promoted the formation of a more stable gel structure in brown fermented milk, improving its storage stability.
Cohesiveness affects gel composition and reflects the intermolecular bonding strength between the components of the sample, thereby influencing its dehydration and shrinkage process. Meanwhile, the index of viscosity objectively reflects the adhesion characteristics of a sample. The cohesiveness of group C samples was higher than that of group S samples, indicating a stronger gel structure and intermolecular interaction in group C and thus preventing quality problems such as whey separation. This indicated that the group C samples would taste smoother and be less prone to stratification during storage, thus maintaining greater homogeneity. The viscosity index of the C group was significantly higher than that of the S group throughout the storage period (p < 0.05), except on D14, where the difference was not significant.

3.4. Probiotic Properties of Brown Fermented Milk During Storage

3.4.1. Determination of VPP and IPP Concentrations

Determining the VPP and IPP content of brown fermented milk is of great significance. Previous studies have shown that these two tripeptides have significant biological activities, including anti-glycemic, cardioprotective, and immunoregulatory effects [24]. VPP and IPP are primarily produced via microbial metabolism. Microbes hydrolyze milk proteins by secreting proteases to produce bioactive peptides with ACE inhibitory activity. As shown in Figure 4A,B, the VPP and IPP contents of group C peaked after 7 days (0.64 ± 0.11 and 2.40 ± 0.29 μmol/L, respectively). During the storage period, the VPP and IPP concentrations of group C remained significantly higher than those of group S (p < 0.05). This difference was primarily due to H11 addition. As shown in Figure 2C, although the viable cell counts were significantly higher in group S than in group C throughout the storage period, this did not impact the VPP and IPP content and benefits of group C samples. The findings indicated that H11 hydrolyzes proteins more efficiently through its protease activity to produce VPP and IPP, demonstrating its significant advantage in the production of bioactive peptides.

3.4.2. ACE Inhibition Rate

ACE inhibitors can reduce blood vessel constriction by preventing the conversion of angiotensin I into angiotensin II, thereby lowering blood pressure and reducing the risk of cardiovascular disease. The storage process affects the ACE inhibitory activity of brown fermented milk via the interaction of multiple factors, such as storage temperature, humidity, oxygen, and microorganisms [25]. This study explored the effect of H11 addition on the ACE inhibitory activity of brown fermented milk while ensuring that the other conditions remain constant. The findings showed that the ACE inhibition rate of group C was significantly higher than that of group S throughout the storage period (p < 0.05), consistent with the results in Section 3.4.1. After 14 days of storage, the ACE inhibition rates peaked in both groups, reaching 67.34 ± 0.24% and 42.65 ± 0.60%, respectively (Figure 4C). In a previous study, H11 demonstrated excellent performance in the production of brown fermented milk beverages, and the highest ACE inhibitory rate was achieved after 21 days (70.13 ± 2.83%) [26]. These findings are consistent with the results of the current study. The research confirms that H11 addition can significantly improve the ACE inhibitory rate of brown fermented milk products.

3.5. Effect of H11 Addition on the Metabolic Changes in Brown Fermented Milk During Storage

Our previous experiments showed that H11 addition can significantly improve the storage stability of brown fermented milk and enhance its ACE inhibitory rate, demonstrating that H11 plays an important role during storage through its unique metabolic pathways and metabolites. To more comprehensively understand the impact of H11 on the quality of brown fermented milk, particularly the mechanism through which H11 improves its biological activity and stability, the metabolome of brown fermented milk was further analyzed. The metabolomes of the two groups of brown fermented milk were detected during storage using UPLC-Q-TOF-MS. First, the stability and reproducibility of the instrument during testing were evaluated. As shown in Figure 5A,B, the repeatability of the QC samples examined before the formal testing period was good, indicating that the instrument was stable and capable of yielding accurate metabolomics data. Throughout the testing process, the results of the QC samples were evenly distributed across the results of all test groups. The final QC samples were clustered together in PCA plots, demonstrating the high accuracy and good repeatability of the testing process, thereby proving the reliability and precision of the protocol. As shown in Figure 5B, there was a clear separation between the samples of groups S and C, indicating that there were differences in the metabolomic characteristics of the two groups. Given that the only variable in the experiment was H11 addition, these results indicated that the separation trend was likely due to H11 addition.
Next, we examined the effect of H11 addition on the metabolomes of brown fermented milk samples. We compared C1-S1, C7-S7, C14-S14, and C21-S21 samples, which were stored for 1, 7, 14, and 21 days, respectively. Differential metabolites were obtained based on the screening criteria VIP > 1.0, FC > 2 or <0.5, and p < 0.05. A total of 21, 11, 21, and 17 differential metabolites were identified after 1, 7, 14, and 21 days of storage, respectively (Figure 5E–H).

3.6. Effects of H11 Addition on Metabolites and Differential Pathway Analysis

Next, we quantified the differential metabolites identified at each time point. As shown in Figure 6A, a total of 26 differential metabolites could only be detected at a specific time point, indicating that H11 addition affected the presence of these metabolites, but this effect was temporary and not persistent. Only two differential metabolites were detected at all four storage time points: pseudouridine (Figure 6G) and shinflavanone (Figure 6F). Further classification of the differential metabolites identified at each time point revealed that most of these metabolites belonged to the following categories: glycerophospholipids, fatty acyls, carboxylic acids and derivatives, organooxygen compounds, and pyridines and derivatives (Figure 6B–E). The pathway analysis of the differential metabolites revealed enrichment for 7, 5, 4, and 4 metabolic pathways at the 1-, 7-, 14-, and 21-day time points, respectively (Figure 7A–D). Among them, 6 metabolic pathways appeared more than once, namely, tyrosine metabolism, vitamin B6 metabolism, tryptophan metabolism, terpenoid backbone biosynthesis, phenylalanine metabolism, and fatty acid biosynthesis. The appearance of these common pathways indicated that H11 addition may affect the overall metabolic profile of brown fermented milk via multiple metabolic pathways, thereby impacting the synthesis and metabolism of flavor substances.

4. Discussion

S. thermophilus and L. bulgaricus usually act synergistically, and their metabolic activities complement each other. During this process, lactose is efficiently converted into lactic acid, promoting the coagulation and formation of milk proteins. Flavor substances are also produced, improving the taste and nutritional value of fermented milk. However, our findings showed that H11 addition can alter this balance. Early in fermentation, the three groups of microbes may compete for resources such as lactose and amino acids, resulting in mutual growth inhibition, which ultimately manifests as a delay in fermentation. As shown in Figure 1A–D, we observed that at the beginning of the fermentation period, the S group (S. thermophilus and L. bulgaricus only) underwent faster fermentation, showing the coagulation of milk proteins and the formation of a network structure. Similarly, Sun et al. [27] found that after the addition of M8, the formation of the gel structure in fermented milk was delayed, consistent with the results of this study. However, as fermentation progressed, the strains in group C gradually established stable growth dynamics, accelerating the fermentation process. The final fermentation duration of group C was 5.85 ± 0.04 h, while that of group S was 6.95 ± 0.05 h. This indicated that group C had a faster fermentation rate, demonstrating that H11 addition can increase the fermentation speed during brown fermented milk production and reduce the fermentation time.
In this study, the fermented milk was stored at 4 °C for 21 days. Low temperatures not only delay the metabolic activity of lactic acid bacteria in brown fermented milk but also inhibit the reproduction of harmful microorganisms. This delay in the acidification of brown fermented milk slows down adverse changes, such as flavor deterioration and whey precipitation. Additionally, the low temperature helps maintain the activity and probiotic effects of bacterial strains such as H11. However, even under low-temperature conditions, the growth and metabolic activity of lactic acid bacteria cannot be completely inhibited. During storage, lactic acid bacteria continue to slowly utilize the nutrients in brown fermented milk to produce acidic substances such as lactic acid, acetic acid, and amino acids. These substances continue to accumulate, gradually increasing the acidity of brown fermented milk and continuously decreasing the pH value, as observed in the present study (Figure 2A,B). Unexpectedly, in this study, a dramatic trend of pH changes was observed in groups C and S. During the storage period, the pH of the two groups did not follow a consistent pattern or appear higher or lower in one specific group Previous studies have shown that pH can change dynamically throughout storage [28,29]. We speculate that the pH fluctuations observed in this study may be due to the interactions between the metabolites of probiotics and the buffering components (milk protein, fat, and minerals) present in fermented milk. At certain time points, the accumulation of acidic substances may have exceeded the buffering capacity of the system, causing a sharp drop in pH. However, as the probiotics continued to produce metabolites, this trend changed again. Ultimately, these factors led to dynamic fluctuations in pH across the two groups.
As brown fermented milk is a probiotic product, measuring its viable cell count is essential. Unfortunately, in this study, the H11 count could not be assessed individually. Hence, the total viable cell count of the samples was ultimately evaluated. There was a significant difference in the viable cell counts of groups C and S during the storage period (p < 0.05), but H11 addition appeared to inhibit the growth of S. thermophilus and L. bulgaricus. This was consistent with the results of TA (Figure 2B). The viable cell count of group S was higher than that of group C, and the acidic substances produced by microorganisms were also more abundant in group S. Therefore, the TA of group S was significantly higher than that of group C throughout the storage period. Wang et al. [30] found that the growth of Bifidobacterium longum in mixed culture with lactic acid bacteria was similar to that in pure culture, but after 48 h of fermentation, the viable cell count was significantly lower than that of the pure culture group. These results show that the addition of strains may not always lead to higher bacterial counts but may instead produce inhibitory effects in some cases. Therefore, the pros and cons of these changes must be evaluated. Nevertheless, in this study, although H11 addition reduced the viable cell counts of the brown fermented milk samples, the C group still had a viable count of 7.175 × 108 CFU/mL at the end of the storage period. This value was much higher than the minimum requirement of 107 CFU/mL for probiotic products [31].
L. helveticus has excellent proteolytic abilities because it produces both intracellular and extracellular proteases [32], which break down the proteins in dairy products and release small peptides and amino acids, including VPP and IPP. In this study, we found that H11 addition significantly increases the contents of VPP and IPP in brown fermented milk, which help regulate blood pressure and exert prebiotic effects. This was attributed to its strong proteolytic ability. Researchers have demonstrated that VPP and IPP can alleviate Angiotensin II-induced vascular dysfunction by modulating the extracellular vesicle-mediated transmission of RNAs between vascular endothelial cells and vascular smooth muscle cells [33]. The isoelectric points of VPP and IPP are approximately 4.79 and 4.49, respectively (https://web.expasy.org/protparam/, accessed on 8 November 2025). Hence, during the storage of brown fermented milk, both these peptides are positively charged. As storage time is prolonged, the bacteria continue to use the nutrients present in dairy products for their growth and metabolism, producing acids and gradually lowering the pH. At low pH levels, VPP and IPP exhibit strong hydrophilicity and thereby show increased solubility. However, as the pH decreases further, VPP and IPP may combine or precipitate along with milk proteins, which reduces their concentration in the solution and ultimately decreases their content in brown fermented milk. The ACE inhibitory rate values observed in this study were consistent with this phenomenon (Figure 4C), indicating that H11 can be successfully applied for the development and production of functional brown fermented milk products, especially those with blood-pressure-lowering effects.
Previous research has shown that H11 addition improves the prebiotic properties of brown fermented milk [34]. Building on these findings, we examined the metabolic characteristics of brown fermented milk produced with and without H11 addition to explore the potential mechanisms underlying the effects of H11. We detected differential metabolites at various storage time points (Figure 5E–H) and found that 18 differential metabolites appeared at multiple stages (Figure 6A). We asked if H11 addition consistently affects the concentrations of these metabolites. The results showed that 16 of the metabolites exhibited consistent changes, with 9 of them being significantly up-regulated and 7 being significantly down-regulated. Further analysis revealed that most of the 9 up-regulated metabolites, including phenylacetaldehyde and phenyllactic acid, were closely related to the flavor of brown fermented milk. Phenyllactic acid is an aromatic compound with a sour taste and distinct aroma. In addition to its antibacterial properties, this compound also increases flavor complexity and enhances the flavor profile of fermented milk [35]. Meanwhile, phenylacetaldehyde is a volatile compound with a strong floral aroma and sweetness and is widely present in fermented foods such as fermented milk and wine. It is considered a key flavor component in various foods and can significantly enhance their aroma and flavor [36]. These findings suggest that H11 addition can not only increase the content of key flavor substances in brown fermented milk but may also enhance its aroma, thereby increasing its consumer appeal.
According to previous studies, H11 exhibits strong proteolytic ability, produces large amounts of VPP and IPP, and demonstrates high ACE inhibitory rates [37]. We attempted to elucidate whether this effect is limited to bioactive peptides. Through further research, we found 3 dipeptides among the differential metabolites detected between groups C and S in this study: asparaginylarginine, prolyl-aspartate, and tryptophyl-lysine. Specifically, H11 addition significantly increased their levels in brown fermented milk. This finding indicates that H11 not only produces biologically active peptides but also significantly alters the levels of other important metabolites in brown fermented milk, potentially having a broader impact on the nutritional properties and functionality of the final product.
Finally, we conducted an in-depth analysis of the metabolic pathways associated with the detected differential metabolites. Two pathways, tyrosine metabolism and vitamin B6 metabolism, were enriched on days 7, 14, and 21, indicating that H11 addition altered the metabolic profile of brown fermented milk. Guo et al. [38] applied Bifidobacterium adolescentis B8589 for the production of probiotic fermented beverages and found that this strain significantly increased the content of beneficial amino acid metabolites and altered the activity of the tyrosine metabolism pathway. Tyrosine metabolism helps prevent allergic respiratory inflammation [39] and is also a key pathway for flavor formation in brown fermented milk. During this process, tyrosine is converted into a range of aromatic compounds, including phenylacetaldehyde and phenyllactic acid [40]. In addition, tyrosine metabolism improves the nutritional value of dairy products by generating vanillic acid, which has antioxidant activity and supports intestinal barrier function [41]. These changes are attributed to the conversion of tyrosine into other beneficial via microbial enzymes such as tyrosine decarboxylase. Overall, our findings showed that H11 addition enhances the activity of the tyrosine metabolism pathway, improving the flavor and probiotic properties of brown fermented milk.
In addition to regulating amino acid metabolism, enzyme activity, and energy balance via vitamin B6 metabolism, H11 also exerts important effects on health and immune function [42]. The vitamin B6 metabolic pathway appeared to be particularly significant during the middle and late stages of brown fermented milk storage, when the accumulation of metabolites gradually influenced microbial metabolism. At this stage, H11 could utilize metabolites such as lactic acid as carbon sources to promote the synthesis and metabolism of vitamin B6. Research shows that Lactiplantibacillus plantarum FMBL L23251 increases vitamin B6 levels in chickpea milk through the 1-deoxy-D-xylulose-5-phosphate (DXP)-independent pathway while also generating a large amount of flavor compounds and eliminating beany odors [43]. In addition, vitamin B6 helps to regulate the balance between sodium and potassium ions, which is essential for maintaining normal blood pressure. When the ratio between these two ions is disrupted, blood pressure elevations can occur. Moreover, the anti-inflammatory and antioxidant effects of vitamin B6 are also crucial for the prevention of hypertension and diabetes [44]. Vitamin B6 deficiency can obstruct glycolysis and gluconeogenesis, leading to glucose metabolism disorders and an increased risk of diabetes [45]. These findings suggest that H11 addition increases amino acid levels in brown fermented milk and may effectively prevent hypertension and diabetes, demonstrating broader market potential for the production of brown probiotic fermented milk.

5. Conclusions

Overall, as an adjunct culture, H11 demonstrates significant value in comprehensively improving the quality of brown fermented milk. It not only optimizes production processes (e.g., by shortening fermentation time) and enhances fundamental qualities (such as texture and flavor), but more importantly, confers well-defined functional properties to the product—notably, antihypertensive potential. Although a slight reduction in total viable cell counts may occur during later storage stages, its advantages in enhancing product stability, delaying post-acidification, and generating functional metabolites are more prominent. These findings provide a solid scientific basis for developing high-quality functional brown fermented milk products and indicate promising prospects for industrial application.

Author Contributions

Conceptualization, W.Z.; methodology, H.C.; software, W.Z. and H.C.; investigation, C.T.; data curation, C.T.; writing—original draft preparation, W.Z.; writing—review and editing, J.L.; visualization, C.T.; supervision, J.L.; funding acquisition, J.L. All authors have read and agreed to the published version of the manuscript.

Funding

The research was funded by the National Natural Science Foundation of China (32202000) and the Inner Mongolia Natural Science Foundation (2021BS03043).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The microrheological indicators of the two groups of brown fermented milk during the fermentation process. (A) Elasticity index (EI). (B) Macroscopic viscosity index (MVI). (C) Solid-liquid balance (SLB). (D) Fluidity index (FI). S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 1. The microrheological indicators of the two groups of brown fermented milk during the fermentation process. (A) Elasticity index (EI). (B) Macroscopic viscosity index (MVI). (C) Solid-liquid balance (SLB). (D) Fluidity index (FI). S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 2. Change in the brown fermented milk over the 21-day storage period (D1, D7, D14, and D21 represent storage for 1, 7, 14, and 21 days, respectively). (A) pH. (B) Titratable acidity (TA). (C) Viable cell counts. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 2. Change in the brown fermented milk over the 21-day storage period (D1, D7, D14, and D21 represent storage for 1, 7, 14, and 21 days, respectively). (A) pH. (B) Titratable acidity (TA). (C) Viable cell counts. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 3. Texture changes in brown fermented milk during storage. (A) Cohesion. (B) Consistency. (C) Hardness. (D) Viscosity index. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 3. Texture changes in brown fermented milk during storage. (A) Cohesion. (B) Consistency. (C) Hardness. (D) Viscosity index. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 4. Changes in VPP, IPP, and ACE inhibition rate among the storage. (A) Represents the VPP concentrations during the 21-day storage. (B) Represents the IPP concentrations during the 21-day storage. (C) Display the ACE inhibition rate. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 4. Changes in VPP, IPP, and ACE inhibition rate among the storage. (A) Represents the VPP concentrations during the 21-day storage. (B) Represents the IPP concentrations during the 21-day storage. (C) Display the ACE inhibition rate. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 5. Changes in the metabolomic profile of brown fermented milk over 21 days of storage. (A) Represent the total ion chromatograms of QC samples, respectively. (B) PCA of the metabolites in all groups. (C) PCA of the metabolites in the C group. (D) PCA of the metabolites in the S group. (E–H) Heatmaps of the differential metabolites at 1, 7, 14, and 21 days of storage. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 5. Changes in the metabolomic profile of brown fermented milk over 21 days of storage. (A) Represent the total ion chromatograms of QC samples, respectively. (B) PCA of the metabolites in all groups. (C) PCA of the metabolites in the C group. (D) PCA of the metabolites in the S group. (E–H) Heatmaps of the differential metabolites at 1, 7, 14, and 21 days of storage. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 6. Differential metabolites identified in brown fermented milk after 1, 7, 14, and 21 days of storage. (A) UpSet analysis of the changes in differential metabolites between the C and S groups. (B–E) Metabolite classification at 1, 7, 14, and 21 days using the HMDB database. (F,G) Amounts of pseudouridine and shinflavanone after 1, 7, 14, and 21 days of storage. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
Figure 6. Differential metabolites identified in brown fermented milk after 1, 7, 14, and 21 days of storage. (A) UpSet analysis of the changes in differential metabolites between the C and S groups. (B–E) Metabolite classification at 1, 7, 14, and 21 days using the HMDB database. (F,G) Amounts of pseudouridine and shinflavanone after 1, 7, 14, and 21 days of storage. Uppercase letters represent inter-group differences, and lowercase letters represent intra-group differences. S: brown fermented milk produced with PYS-010 alone, C: brown fermented milk produced with a combination of PYS-010 and H11.
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Figure 7. Enrichment analysis of differential metabolites. (A) Metabolic pathway analysis at day 1 of storage. (B) Metabolic pathway analysis at day 7 of storage. (C) Metabolic pathway analysis at day 14 of storage (D) Metabolic pathway analysis at day 21 of storage.
Figure 7. Enrichment analysis of differential metabolites. (A) Metabolic pathway analysis at day 1 of storage. (B) Metabolic pathway analysis at day 7 of storage. (C) Metabolic pathway analysis at day 14 of storage (D) Metabolic pathway analysis at day 21 of storage.
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MDPI and ACS Style

Zhai, W.; Tian, C.; Chen, H.; Li, J. Effects of Lactobacillus helveticus H11 Addition on the Fermentation, Storage, and Probiotic Properties of Brown Fermented Milk. Foods 2026, 15, 606. https://doi.org/10.3390/foods15040606

AMA Style

Zhai W, Tian C, Chen H, Li J. Effects of Lactobacillus helveticus H11 Addition on the Fermentation, Storage, and Probiotic Properties of Brown Fermented Milk. Foods. 2026; 15(4):606. https://doi.org/10.3390/foods15040606

Chicago/Turabian Style

Zhai, Wenkang, Chunle Tian, Huimin Chen, and Jianli Li. 2026. "Effects of Lactobacillus helveticus H11 Addition on the Fermentation, Storage, and Probiotic Properties of Brown Fermented Milk" Foods 15, no. 4: 606. https://doi.org/10.3390/foods15040606

APA Style

Zhai, W., Tian, C., Chen, H., & Li, J. (2026). Effects of Lactobacillus helveticus H11 Addition on the Fermentation, Storage, and Probiotic Properties of Brown Fermented Milk. Foods, 15(4), 606. https://doi.org/10.3390/foods15040606

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