Probiotic Bifidobacterium animalis ssp. lactis Probio-M8 improves the fermentation and probiotic properties of fermented milk

Probiotics are increasingly used as starter cultures to produce fermented dairy products; however, few studies have investigated the role of probiotics in milk fermentation metabolism. The current study aimed to investigate whether adding Bifidobacterium animalis ssp. lactis Probio-M8 (Probio-M8) as a starter culture strain could improve milk fermentation by comparing the physico-chemical characteristics and metabolomes of fermented milks produced by a commercial starter culture with and without Probio-M8. Our results showed that adding Probio-M8 shortened the milk fermentation time and improved the fermented milk texture and stability. Metabolomics analyses revealed that adding Probio-M8 affected mostly organic acid, AA, and fatty acid metabolism in milk fermentation. Targeted quantitative analyses revealed significant increases in various metabolites related to the sensory quality, nutritive value, and health benefits of the probiotic fermented milk, including 5 organic acids (acetic acid, lactic acid, citric acid, succinic acid, and tartaric acid), 5 EAA (valine, arginine, leucine, isoleucine, and lysine), glutamic acid, and 2 essential fatty acids (α-linolenic acid and docosahexaenoic acid). Thus, applying probiotics in milk fermentation is desirable. This study has generated useful information for developing novel functional dairy products.


INTRODUCTION
Probiotics are sometimes added to foods for their health-promoting effects.Adequate amounts of active probiotics should be ingested to confer beneficial functions (Guo et al., 2022).However, the survival and activities of probiotics may be affected by the food matrix and food processing procedures (Sakandar and Zhang, 2021).Milk is a common carrier of probiotics, and there is a long history (over 5,000 yr) of human consumption of dairy and fermented dairy products, particularly those fermented by lactic acid bacteria (Sakandar and Zhang, 2022).Fermented milk is considered one of the best choices for functional food worldwide.Probiotics can be implemented in fermented milk production processes, and in fact, optimized fermentation favors the survival of lactic acid bacteria (Sakandar and Zhang, 2021).
In recent years, there has been multidimensional research on probiotic fermented milk, ranging from basic science to industrial applications.Increasing evidence suggests that applying probiotics as components of the starter culture in milk fermentation not only improves the product quality and fermentation process but also enhances the probiotic effects of the products (Wang et al., 2021a;FitzGerald et al., 2022).For example, a bifidobacterial-containing probiotic fermented milk has been shown to improve gastrointestinal function and alleviate constipation, possibly due to its increased efficiency in producing short-chain fatty acids (Wang et al., 2020).Probiotic milk metabolites, such as γ-aminobutyric acid and bioactive peptides (e.g., tryptophan-tyrosine and tryptophan-methionine), have been shown to contribute to improving sleep quality and preventing cognitive disorders (Sakandar and Zhang, 2021).Furthermore, daily consumption of probiotic fermented milk could strengthen immunity against influenza viruses (Takahashi et al., 2019).Another factor influencing consumer choices is product features, especially sensory quality.Probiotic application in milk fermentation could improve the fermented milk metabolomics profile, enriching desired volatile metabolites that impart the characteristic taste and flavor of fermented milk, such as acetaldehyde, diacetyl, and medium-chain fatty acids (Wang et al., 2021a).Moreover, it also increases exopolysaccharides (EPS) production, improving the texture quality and viscosity of fermented milk (Bai et al., 2020).
Fermented milk quality is affected by the choice of starter culture strains.Their metabolic activities not only affect the rheological and sensory properties of the product but also determine the potential health benefits of the functional product (Mituniewicz-Malek et al., 2017).Bifidobacterium animalis ssp.lactis BB-12 is often used in fermented dairy products because it is a stable, active, and highly viable strain in the milk fermentation process, and its addition can improve the rheological properties and sensory quality of the resultant fermented milk (Nocerino et al., 2020).Bifidobacterium animalis ssp.lactis Probio-M8 (Probio-M8) is a novel strain isolated from a healthy woman's breast milk in 2017 (Liu et al., 2020).It is a potential strain showing good probiotic characteristics, including acid and bile tolerance and adhesive properties.It also offers various probiotic functions after consumption, such as maintaining gut homeostasis, regulating bone metabolic balance, improving asthma, and easing anxiety and depression (Ma et al., 2023).
Because Probio-M8 is a member of the same subspecies as Bifidobacterium animalis ssp.lactis BB-12, it may have similar fermentation properties, sensory characteristics, and functional effects in milk fermentation.However, as these properties vary between strains, it is necessary to evaluate the fermentation characteristics of each of them.The current study aimed to comprehensively investigate whether implementing Probio-M8 could improve milk fermentation by comparing the physicochemical characteristics and metabolomes of fermented milks produced by a commercial starter culture with and without Probio-M8.The physicochemical characteristics and metabolomes were monitored by rheological characteristics, texture, survival rate of probiotics, water retention, and nontargeted metabolomics, targeted quantification, respectively.This study has provided interesting information on the role of probiotics in the milk fermentation process, and theoretical support and validation targets for the development of Probio-M8 fermented milk in animal and human experiments.

Fermented Milk Production and Sampling
The milk sterilized at 139°C for 2 s was purchased from Shepherd (Beijing) Dairy Development Co. Ltd.The nutrient contents of the milk are shown in Table 1.The probiotic Probio-M8 strain was purchased from Jinhua Yinhe Biological Science and Technology Co. Ltd. (Jinhua, Zhejiang, China), and the commercial starter culture, YoFlex Premium 1.0 (containing Streptococcus thermophilus and Lactobacillus delbrueckii ssp.Bulgaricus), was purchased from CHR-Hansen Holding (Hørsholm, Denmark).
The milk (96%, vol/vol) was preheated to 60°C in a water bath, and sucrose (4%, wt/vol) was added and stirred for 15 min until completely dissolved.The mixture was homogenized (63°C, 20 MPa) in a high-pressure homogenizer (APV Model 2000, SPX Corp., Charlotte, NC).The homogenized milk was then pasteurized at 85°C for 30 min, and rapidly cooled to 37°C (Sun et al., 2023a) before starter culture inoculation and fermentation.The milk was fermented by using either the commercial starter culture alone (0.02 g/1,000 g [3.0 × 10 6 cfu/mL]; FM) or the same dose of commercial starter culture together with 5.0 × 10 7 cfu/mL of Probio-M8 (FM8).The study design is illustrated in Figure 1.
Milk fermentation was performed in 250-mL blue-cap bottles containing 200 mL of inoculated milk at 42°C until reaching the pH of 4.5 when fermentation was terminated by cooling to 20°C in a water bath.Samples were collected every hour during the fermentation process.All experiments were performed in triplicates.

Rheological Measurements
Gel formation during milk fermentation was evaluated by multispeckle diffusing wave spectroscopy (MS-DWS) using a Rheolaser Master optical micro-rheology analyzer (Formulaction, Toulouse, France).Briefly, milk samples (20 mL) were poured into glass tubes (inner diameter of 27.5 mm for the Rheolaser Master) before being placed in the measurement cell and equilibrated at 42°C.Micro-rheological indicators, including elastic index (EI), fluidity index (FI), macroscopic viscosity index (MVI), and solid-liquid balance (SLB), were measured and calculated every 5 min using the built-in software until the sample reached pH 4.5.The data of rheological measurements were statistically analyzed also by the built-in software of the Rheolaser Master Optical micro-rheology analyzer.

pH Value and Titratable Acidity
The pH of the fermented milk samples was determined using a pH meter (pH FE20, Mettler Toledo, Greifensee, Switzerland).The titratable acidity (TA; °T) was deter- Pasteurized milk was fermented at 42°C by using either a commercial starter culture (SC) alone (FM group) or the same dose of SC with Bifidobacterium animalis ssp.lactis Probio-M8 (FM8 group).Samples were collected when the pH reached 4.5.The pH, titratable acidity (TA), viable cell count, content of extracellular polysaccharide substances (EPS), water holding capacity (WHC), and texture and rheological features of the fermented milk were measured.The milk metabolomes were detected by LC-MS/MS, and organic acid, AA, and fatty acid contents were quantified by UPLC-MS/MS and GC-MS.Finally, metabolic pathway enrichment analysis was performed on identified differential metabolites between the 2 types of fermented milks.mined by mixing 5 g of fermented milk with 40 mL of boiled and cooled distilled water, followed by titrating with 0.1 N NaOH using 0.5% phenolphthalein indicator.(Sun et al., 2023a).

Viable Cell Count
Fermented milk samples (25 g) were mixed with 225 mL of sterile PBS (8 g of NaCl/L, 0.2 g of KH 2 PO 4 /L, and 1.15 g of Na 2 HPO 4 /L; pH 7.2) in a sterilized triangular flask at low temperature for 15 min.A suitable amount of diluted fermented milk was applied to De Man, Rogosa, and Sharp agar containing 0.05% l-cysteine and 0.05 mg/mL of mupirocin to enumerate the viable count of Probio-M8 (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, China; Liu et al., 2020).

Viscosity Analysis
The viscosity of fermented milk samples (at 25°C) was measured using a Brookfield viscometer (Model DV2T, Brookfield Engineering Laboratories Inc., Middleboro, MA).Fermented milk samples were measured with stirring during a 30-s continuous scan using a 60 rpm of spindle No. 4 (Guo et al., 2021).

Water Holding Capacity
Fermented milk samples (20 g) were placed in a funnel containing a filter paper for 120 min at 4°C.The filtrate was collected and weighed.The value of water holding capacity (WHC) = (1 − weight of filtrate/weight of fermented milk) × 100% (Guo et al., 2021).

Texture Analysis
Fermented milk samples (80 mL; 20°C −25°C) were analyzed in a single compression cycle test using a texture analyzer TA-XT Plus (Stable Micro Systems Ltd., Godalming, Surrey, UK) with a 5-kg load cell.The test speed was fixed at 1 mm/s, and the penetration depth was 30 mm.Force-time curves were plotted using Texture Expert (Stable Micro Systems Ltd., Godalming, Surrey, UK), and the parameters of hardness, consistency, and viscosity were recorded (Bai et al., 2020).

Extraction and Quantification of EPS
The extracellular polysaccharide substances (EPS) extraction method was described by Bai et al. (2020).In short, 100 mL of fermented milk was centrifuged twice at 4°C (10,000 × g, 30 min).The supernatants were mixed with cold anhydrous ethanol until EPS was precipitated.Afterward, the mixture was centrifuged again at 4°C (10,000 × g, 30 min), ultrasonicated for 2 h (60 kHz, 100 W), and then dialyzed in distilled water for 72 h.The EPS was quantified using the phenol-sulfuric acid method and expressed as glucose equivalent (Bai et al., 2020).The quantity of EPS in samples was determined by interpolation from a preconstructed glucose standard curve spanning 40 to 400 mg/L glucose.

Milk Metabolomics by LC-MS/MS
Fermented milk samples (100 μL) were vortex mixed with 400 μL of the extraction solution (methanol: acetonitrile = 1:1, V/V) for 30 s in an Eppendorf tube, sonicated for 10 min in an ice-water bath, and allowed to stand at −40°C for 1 h.After centrifugation for 15 min at 4°C (13,800 × g, radius = 8.6 cm), the supernatants were transferred to fresh sample vials for liquid chromatography-tandem MS (LC-MS/MS) analysis.An equal amount of all samples was mixed to composite the quality control sample to be run to ensure the stability of the instrumental and chromatographic conditions.
The LC-MS/MS analysis was performed using an ultra-HPLC system (Vanquish; Thermo Fisher Scientific, Waltham, MA) with an ultra-performance liquid chromatography (UPLC) BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS; Thermo Fisher Scientific, Waltham, MA).The liquid chromatography phases were an aqueous phase A, containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia water; phase B was acetonitrile.The gradient elution was programmed as follows: 0 to 0.5 min, 95% B; 0.5 to 7 min, 95% to 65% B; 7 to 8 min, 65% to 40% B; 8 to 9 min, 40% B; 9 to 9.1 min, 40% to 95% B; 9.1 to 12 min, 95% B. The sample pan temperature was 4°C, and the injection volume was 2 μL.The Q Exactive HFX mass spectrometer was used to acquire MS/MS spectra on the information-dependent acquisition mode using an acquisition software (Xcalibur, Thermo).The electrospray ionization source conditions were as follows: sheath gas flow rate of 3.35 L/min; aux gas flow rate of 16.8 L/min; capillary temperature of 350°C; mass range of 70 to 1,050; scan/cycle time of 760 ms; full MS resolution of 60,000; MS/MS resolution of 7,500; collision energy of 10/30/60 in normalized collision energy mode; spray voltage of 3.6 kV (positive ion mode) or 3.2 kV (negative ion mode), respectively.The original data file was converted by ProteoWizard software to mzXML format.

Quantification of Organic Acids and AA by UPLC-MS/MS
Fermented milk samples (0.1 mL) were mixed with 2.4 mL of distilled water and centrifuged at 12,000 × g for 10 min at 4°C.The supernatants (10 μL) were collected, The organic acids and AA were analyzed by UPLC-MS/MS (Sun et al., 2023a).An Acquity UPLC system (AB Sciex, Framingham, MA) was coupled to a triple quadrupole mass spectrometer (AB Sciex, Framingham, MA).Chromatographic separations were performed on a Phenomenex column (2.6-μm EVO C18 100A 2.1 × 100 mm; Torrance, CA).The flow rate was 0.4 mL/min.The mobile phase was composed of water-0.1% formic acid (A) and acetonitrile-0.1% formic acid (B).The temperature of the column was maintained at 40°C, and the injection volume was 2 μL.Mass spectrometric scans were obtained using an electrospray ion source and multiple reaction monitoring mode.

Quantification of Fatty Acids by GC-MS
Fermented milk samples (100 g) were mixed with 2 mL of methyl nonadecylate (Sigma-Aldrich, St. Louis, MO), 100 mg of pyrogallic acid, 2 mL of 95% ethanol, 5 mL of ammonia, and 4 mL of distilled water.The mixed samples were kept in a water bath for 20 min (70°C to 80°C), then cooled to room temperature, mixed with 10 mL of 95% ethanol, transferred to the separation funnel.The flask and plug were washed with 50 mL of ether petroleum ether mixture.The washing solution was added to the separator funnel, covered, shaken for 5 min and left for 10 min, before the ether layer extract was collected.The above steps were repeated to extract the hydrolysates 3 times.The extracted hydrolysates from each sample were pooled, concentrated, and dried until the oil droplets disappeared.After cooling the samples, 20 mL of n-heptane was mixed and shaken for 2 min, before adding saturated sodium chloride solution.The mixtures were allowed to stand for stratification.For each sample, 5 mL of n-heptane extract was mixed with 5 g of anhydrous sodium sulfate, and the mixtures were allowed to stand for 5 min.The upper n-heptane layers containing the extracted fatty acids were transferred into fresh sample vials for GC-MS analysis.
Fatty acids were analyzed using GC-MS (Tra-ce1310ISQ, Thermo Fisher Scientific, Waltham, MA) using the following parameter settings (Qamar et al., 2019): phase column, TG-5MS (30 m × 0.25 mm × 0.25 μm; Thermo Fisher Scientific, Shanghai, China); temperature program: 80°C for 1 min, raised to 200°C at 10°C/ min, 250°C at 5°C/min, 270°C at 2°C/min, and held for 3 min; sampler temperature was 290°C; carrier gas flow rate was 1.2 mL/min; and unsplit stream sampling was used with an opening time for valve of 1 min.The MS conditions were as follows: ionization temperature of 280°C; transmission line temperature of 280°C; solvent delay time of 5 min; scan area of 30 to 400 atomic mass units; and ionization voltage of 70 eV.

Statistical Analysis
The data for pH, TA, viable count, WHC, viscosity, texture, EPS, and target metabolite quantities were evaluated using ANOVA with a cutoff confidence level of 95%.Fermented milk metabolomes were analyzed by principal component analysis (PCA) and orthogonal partial leastsquares discriminant analysis (OPLS-DA).Significantly differential metabolite features were defined by the cutoff level of variable importance on projection (VIP >1) and P-value (P < 0.05).Metabolic pathways annotation and enrichment analysis of differential metabolites were performed by the online tool, Metaboanalyst 5.0 (https: / / www .metaboanalyst.ca).Results were visualized using R packages version 4.0.4 and Origin 2019.Data are presented as mean ± SD unless otherwise stated.

Effect of Probio-M8 on Fermented Milk Rheological Characteristics
A Rheolaser Master instrument was used to analyze the rheological characteristics (SLB, EI, FI, and MVI) of the gel process of the probiotic and nonprobiotic fermented milks (FM8 and FM, respectively).At the end of fermentation, the SLB value was 0.6 for FM8 (versus 0.5 for FM), suggesting a slightly more viscous liquid state in FM8 than FM (Figure 2a).The EI reflects the elastic properties of samples; the higher the EI, the stronger the elasticity and gel structure stability.The MVI is used to reflect the viscosity characteristics of the sample at the micron scale.Both the EI and MVI values of FM8 increased significantly after 3.5 h of fermentation, which occurred slightly earlier than FM (Figure 2b, 2c).The values of both indicators were obviously higher in FM8 than FM, and the differences remained at the end of the fermentation process (P < 0.05).These results suggested that the gel structure of FM8 was more elastic and stable than that of FM.The FI reflects the speed of movement of particles in the fermented milk.It showed an opposite trend compared with EI and MVI.During early fermentation, the FI values of both fermented milks fluctuated at higher levels (ranging between 100 and 1,000 Hz), followed by a sharp drop after 3.5 h of fermentation in FM8 (versus 4 h in FM; Figure 2d).However, at the fermentation endpoint, the value of FI was not significantly higher in FM8 than FM (both at around 10 −2 Hz).These results indicated that FM8 was slightly more viscous than FM and had a more stable gel structure at the fermentation endpoint.

Effect of Probio-M8 on Fermented Milk pH Value, TA, Viable Cell Count, Viscosity, WHC, and EPS Production
Both fermented milks showed a decreasing trend in pH and increasing trend in water holding activity (Figure 3a  and 3b).However, compared with FM, FM8 required a significantly shorter time to reach the fermentation endpoint (5.5 h for FM8 vs. 6 h for FM; P = 0.0152; Figure 3a), and had a significantly higher WHC at the fermentation endpoint (50.34% for FM8 vs. 47.16% for FM; P = 0.0027; Figure 3b).The FM8 group was significantly more viscous than FM (1.27-time difference; P = 0.0027; Figure 3c), which was likely because EPS production was not significantly higher (Figure 3d).At the fermentation endpoint, the TA level of FM8 was not significantly higher than that of FM (Figure 3e).The Probio-M8 count in FM8 was 3.4-time higher at the fermentation endpoint compared with the initial count at baseline (1.7 × 10 8 cfu/ mL vs. 5.0 × 10 7 cfu/mL; Figure 3f).

Effect of Probio-M8 on Fermented Milk Texture
We compared the texture characteristics (namely cohesiveness, consistency, viscosity, and hardness) of the probiotic and nonprobiotic fermented milks at the fermentation endpoint.Compared with FM, FM8 had significantly higher cohesiveness, consistency, and viscosity levels (Figure 4a, 4b, 4c; P < 0.05).However, no significant difference was seen in hardness between the 2 fermented milks (Figure 4d).These results suggested that adding Probio-M8 could increase the viscosity of the fermented milk, making it more stable.

Effect of Probio-M8 on Fermented Milk Metabolomes
Metabolomics analysis was applied to investigate the changes in milk composition after fermentation with and without adding Probio-M8.First, a PCA was performed.On the PCA score plot, sample type-clustering pattern was observed (Figure 5a), suggesting obvious metabo- lomic differences between sample types.A total of 805 metabolites were detected; and the top 2 metabolite classes were: lipids and lipid-like molecules (27.8%) and organic acids and derivatives, respectively (20.57%, Figure 5b).Glycerophospholipids (14.04%) and fatty acids (8.51%) were the 2 largest metabolite subclasses within the metabolite class, lipids and lipid-like molecules, while carboxylic acids and derivatives (15.74%) was the largest metabolite subclass within the metabolite class organic acids and derivatives (Figure 5b).Qualitative matching analysis identified 705 metabolites, and around half (48.37%) of these metabolites belonged to the metabolite classes of organic acids and derivatives (145 metabolites) and lipids and lipid-like molecules (196 metabolites), respectively.Our further analysis thus focused on these 2 metabolite classes.
We then performed an OPLS-DA of the metabolomics (comprising only organic acids and derivatives, lipids, and lipid-like molecules) between the 2 fermented milks (Figure 5c).The OPLS-DA is based on supervised classification.By filtering out orthogonal variables that are irrelevant to metabolic variable classification, differential metabolites between groups of samples could be reliably spotted.Our OPLS-DA model was of good performance, evident by the validity indicators of effectiveness (R 2 Y = 0.862) and accuracy (Q 2 = 0.685).On the OPLS-DA score plot, the symbols representing FM8 and FM showed distinct group-based clustering pattern, suggesting that adding Probio-M8 caused obvious changes in the metabolite classes of organic acids and derivatives, and lipids and lipid-like molecules in fermented milk.The OPLS-DA analysis further revealed a total of 78 differential metabolites between FM8 and FM (cutoff threshold: VIP >1 and P < 0.05; Supplemental Table S1, see Notes), including 38 lipids and lipid-like molecules and 41 organic acids and derivatives.Metabolic pathway analysis of these metabolites identified 25 enriched metabolic pathways (Figure 5d), which were related: fatty acid metabolism (2 pathways), organic acid metabolism (5 pathways), and AA metabolism (18 pathways).
These results suggested that adding Probio-M8 affected mainly metabolites and metabolic pathways related to the metabolism of fatty acids, organic acids and amino acids.Therefore, we focused on quantifying selected metabolites of these 3 groups of macromolecules.
The results of quantification of organic acids, AA, and fatty acids were analyzed by OPLS-DA (Figure 6a, 6b, and 6c, respectively; 6a: R 2 Y = 0.999, Q 2 = 0.997; 6b: R 2 Y = 0.971, Q 2 = 0.914; 6c: R 2 Y = 0.995, Q 2 = 0.985; R 2 Y represents the interpretation ability of the model, and Q 2 represents its predictive ability).In all 3 OPLS-DA score plots, clear sample-based clustering pattern was observed, suggesting obvious overall differences in these 3 groups of macromolecules between samples.The quantification data were also visualized in a hierarchical clustering heat map (Figure 6d).Each set of triplicate  samples shared a highly similar metabolite distribution pattern, indicating a high accurate and stability in the metabolite quantification techniques and experimental conditions.Notably, many of the quantified macromolecules (including tartaric acid, acetic acid, citric acid, succinic acid, ALA, DHA, threonine, glutamic acid, valine, leucine, lysine, and arginine; P < 0.05) were significantly more abundant in FM8 than FM, although few of them showed an opposite trend (including malic acid, capric acid, and phenylalanine; P < 0.05; Figure 6e, 6f, and 6g).
The unfermented milk contained the least amounts of the quantified macromolecules in most cases.This is an interesting result suggesting that the milk fermentation process could release many of these metabolites, while adding Probio-M8 as a starter culture strain could further enhance their production in the fermentation process.

DISCUSSION
Probiotics are increasingly used to produce fermented dairy products; however, few studies have investigated the role of probiotics in milk fermentation metabolism, particularly its relation with the physicochemical characteristics of the final products.Thus, this study investigated the effects of adding Probio-M8 on the fermented milk features and metabolomes, focusing on the compositional differences in organic acids, AA, and fatty acids contents between the probiotic and nonprobiotic fermented milk.
The growth and survival ability of bacteria is a fundamental criterion for evaluating the functional potential of probiotic foods (Guo et al., 2022).To effectively confer health-promoting function, probiotic fermented milk should contain a minimal probiotic viable count exceeding 10 6 cfu/mL during the product shelf life (Bai et al., 2020).The probiotic viable count of Probio-M8 in FM8 was 1.71 × 10 8 cfu/mL, fulfilling such requirement.
The choice of starter cultures may affect the milk fermentation process and the product characteristics, such as texture and stability (Bai et al., 2020).Adding Probio-M8 could improve the quality and stability of the fermented milk by shortening fermentation time, increasing viscosity, and enhancing the elastic gel system.The active organic acid metabolism of Probio-M8 may have caused an increased acid production rate, leading to a shorter fermentation time.In FM8, more EPS was detected, which may account for the increased viscosity.These metabolic changes can help form a dense protein network made up of casein (Guo et al., 2022), influencing the flavor, texture, and functional properties of fermented milk (Sun et al., 2023a).
To further investigate the metabolic basis of our observations, we performed comparative milk and fermented milk metabolomics analyses.Many differential metabolites between FM8 and FM belonged to 2 metabolite classes, namely organic acids and derivatives and lipids and lipid-like molecules.Further metabolic pathway analysis of these metabolites identified 25 enriched metabolic pathways involved in fatty acid, organic acid, and AA metabolism.Based on the differential metabolites (6 organic acids, 9 AA, and 6 fatty acids) and enriched metabolic pathways, we constructed a metabolic pathway map highlighting the effect of Probio-M8 addition on the milk metabolome (Figure 7; Wang et al., 2021b;Sun et al., 2023a).
Carboxylic acids are the most common organic acids.In our dataset, carboxylic acids account for about three-quarters of total organic acid metabolites.Shortchain carboxylic acids are important chemical factors that influence the flavor of dairy products.These acids mainly originate from AA metabolism, tricarboxylic acid (TCA) cycle conversion, and the Embden-Meyerhof-Parnas pathway (Sun et al., 2023b).It is worth noting that bifidobacteria have a unique glycolytic pathway, namely the hexokinase pathway (Guo et al., 2022).In the hexokinase pathway, glucose is phosphorylated to glucose-6-phosphate, which is eventually converted to acetic acid and lactic acid through the actions of multiple enzymes.This explains why FM8 has more acetic acid and lactic acid than FM.Lactic acid plays a role in regulating the TCA cycle (Hui et al., 2017), which is prone to convert lactic acid into other organic acids when its cellular level is high, helping to maintain the cellular lactic acid concentration within an acceptable range.The TCA cycle is also involved in energy generation, coenzyme regeneration, supply of metabolic intermediates, and imparting flavor characteristics to fermented milk (Martínez-Reyes and Chandel, 2020).Our study found that adding Probio-M8 could variably modulate some TCA cycle metabolites (e.g., enhancing the release of citric acid and succinic acid, while decreasing malic acid).Citric acid is the starting material of the TCA cycle, which was more abundant in FM8 than FM.It imparts a refreshing and bright flavor to fermented dairy products (Lu et al., 2018).Succinic acid is a signaling molecule that regulates cellular oxidative stress and inflammatory responses (Sun et al., 2023b).Malic acid adds a light and fruity flavor to fermented milk (Marques et al., 2020).The FM8 group has significantly less malic acid, possibly due to its conversion to tartaric acid; this finding is consistent with the increased quantity of tartaric acid in FM8 compared with FM (144.64 mg/kg vs. 119.61mg/ kg, respectively).Tartaric acid provides a fruity flavor to fermented milk and inhibits some environmental bacteria (Wang et al., 2021b).Thus, Probio-M8 could alter the organic acid metabolism in the milk fermentation process, increasing the concentrations of acetic acid, lactic acid, citric acid, succinic acid, and tartaric acid, thus improving the sensory characteristics of fermented milk.Bacteria in the milk matrix can easily affect AA metabolism during milk fermentation (Le and Yang, 2022).Amino acids can influence the taste of fermented milk, as they are the precursors of many flavor compounds, such as alcohols, aldehydes, and esters (Zhang et al., 2017).The TCA cycle and bacterial protein degradation of raw milk are the primary sources of the amino acids in fermented milk.Amino acid metabolism generates metabolic intermediates that enter the TCA cycle (Zhao et al., 2020).For example, valine and arginine are precursors of α-ketoglutarate, a key intermediate in the TCA cycle.
Our study found that FM8 has more valine and arginine than FM.Valine, leucine, and isoleucine are collectively known as branched-chain AA.These AA can improve exercise performance and alleviate premature ovarian failure (Yan et al., 2022;Guo et al., 2023).However, a high level of isoleucine is unfavorable for longevity (Weaver et al., 2023).The FM8 group has significantly more valine and leucine than FM, but the isoleucine level is identical in both fermented milks (0.12 mg/100 g).Furthermore, branched-chain AA can enhance the growth of probiotic bacteria by regulating metabolic pathways, such as pu-rine biosynthesis and AA synthesis (Le and Yang, 2022).The FM8 group also has significantly more lysine and threonine, which can protect bacterial cell structure and maintain normal physiological functions (Le and Yang, 2022).The increases in the cellular concentrations of the aforementioned AA could help sustain the viability of Probio-M8 at the fermentation endpoint.Additionally, adding Probio-M8 could substantially promote glutamate production; glutamate is a crucial metabolite connecting the TCA cycle, AA metabolism, and the γ-aminobutyric acid pathway (Sun et al., 2023a).Glutamate is a NEAA and the most abundant AA in the human body.It plays a critical role in the digestive and immune systems and is a precursor for some neurotransmitters (Liang et al., 2023).In contrast, significantly less phenylalanine was detected in FM8 than in FM.In humans, phenylalanine accumulation is detrimental to health, and it is typically converted to tyrosine to prevent phenylketonuria (Rondanelli et al., 2023).Our results showed that adding Probio-M8 as a starter culture could produce more amino acids (including 5 EAA, namely valine, arginine, leucine, isoleucine, and lysine), which is desirable.Fatty acid metabolism is connected to the TCA cycle through the substrate acetyl coenzyme A (Guo et al., 2022).Fatty acids are associated with fermented milk texture, flavor, mouthfeel, and health benefits.Unsaturated fatty acids are classified into MUFA and PUFA based on their double bonds.Oleic acid is the main UFA found in fermented milk.Oleic acid can regulate blood lipids and reduce excessive inflammatory responses in critically ill patients (Wang et al., 2022).Our study showed that FM8 has less oleic acid compared with FM, which is in line with the results reported by previous works (Li et al., 2020;Zha et al., 2021;Sun et al., 2023b).The reduction in oleic acid is attributed to the probiotic-driven conversion of oleic acid to stearic acid through hydrogenation (Sun et al., 2023b), as confirmed by the higher amount of steric acid in FM8 than in FM (Appendix Figure A1).Representative PUFA include ALA, DHA (n-3 family), and ARA (n-6 family).The synthesis of ALA is limited in mammals because of the lack of desaturases responsible for introducing unsaturated double bonds (Guo et al., 2022).Increasing dietary intake of ALA may bring additional health benefits, such as reducing cardiovascular disease and fatal coronary heart disease risks and mitigating cognitive impairment (Bernasconi et al., 2021;Sala-Vila et al., 2022;Keenan et al., 2023).Both ARA and DHA are converted from ALA.The significant increases in ALA and DHA in FM8 may cause a decrease in C4-C10 fatty acids (Guo et al., 2022).The FM8 group has less decanoic acid than FM, but nonsignificantly more butyric acid.Butyric acid is a beneficial short-chain fatty acid that regulates metabolism, reduces inflammation, and enhances immunity (van Dueren et al., 2022).Adding Probio-M8 as a starter culture could increase essential fatty acids, including ALA and DHA, making FM8 a better functional fermented milk.

CONCLUSIONS
This study found that adding Probio-M8 could speed up the milk fermentation process and improve the physicochemical and functional properties of fermented milk.Metabolomics analyses revealed that Probio-M8 exerts a substantial effect on the organic acid, fatty acid, and AA metabolism in milk fermentation, increasing the concentrations of various metabolites relating to the sensory quality, nutritive value, and health benefits, including 5 organic acids (acetic acid, lactic acid, citric acid, succinic acid, and tartaric acid), 5 EAA (valine, arginine, leucine, isoleucine, and lysine), glutamic acid, and 2 fatty acids (ALA and DHA).Therefore, adding probiotics as a starter culture strain in milk fermentation enhanced the desired features of the final products.

NOTES
This study was funded by the Research Fund for the National Key R&D Program of China (Beijing, China; 2022YFD2100700), the earmarked fund for CARS (Beijing,China;, and the Inner Mongolia Science and Technology Major Projects (Hohhot, China; 2021ZD0014).Supplemental material for this article is available at https: / / doi .org/ 10 .6084/m9 .figshare.26114212.v1.Author contributions included Yaru Sun: writing-original draft, investigation, validation, visualization; Shuai Guo: writing-original draft, investigation, validation; Lai-Yu Kwok: writing-review and editing; Zhihong Sun: conceptualization, investigation, writing-review and editing; Jicheng Wang: conceptualization, investigation, writing-review and editing; and Heping Zhang: conceptualization, supervision, project administration.No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.The authors have not stated any conflicts of interest.

Figure 3 .
Figure 3. Effects of Bifidobacterium animalis ssp.lactis Probio-M8 on physicochemical characteristics of fermented milk.(a) pH.The inset represents fermentation time required to reach the fermentation endpoint pH 4.5 (FT).(b) Water holding capacity.The inset represents water holding capacity at fermentation endpoint (pH 4.5).(c) Viscosity; (d) content of extracellular polysaccharides (EPS); (e) titratable acid; (f) probiotic viable count at baseline (FI) and fermentation endpoint (FT).The FM and FM8 groups were the probiotic and nonprobiotic fermented milks, fermented with the commercial starter culture, YoFlex Premium 1.0, with and without Bifidobacterium animalis ssp.lactis Probio-M8, respectively.Error bars represent SD. ns: no significant difference, ANOVA at the 95% confidence level.

Figure 4 .
Figure 4. Texture parameters of milk fermented with and without probiotics.(a) Cohesiveness; (b) consistency; (c) viscosity; (d) hardness.Error bars represent SD. ns = no significant difference, ANOVA at the 95% confidence level.The FM and FM8 groups were the probiotic and nonprobiotic fermented milks, fermented with the commercial starter culture, YoFlex Premium 1.0, with and without Bifidobacterium animalis ssp.lactis Probio-M8, respectively.

Figure 5 .
Figure 5. Effects of adding Bifidobacterium animalis ssp.lactis Probio-M8 on the fermented milk metabolomes.(a) Principal component (PC) analysis score plot of milk metabolomes of probiotic and nonprobiotic fermented milks (FM and FM8, fermented with the commercial starter culture, YoFlex Premium 1.0, with and without Bifidobacterium animalis ssp.lactis Probio-M8, respectively).M represents the prefermented milk.(b) Donut plot shows the distribution of metabolite classes.Super class (inner circles) and class (outer circle) represent the secondary and tertiary classification of metabolites in the Human Metabolome Database (https: / / hmdb .ca/), respectively.(c) Orthogonal partial least squares-discriminant analysis score plots of milk metabolomes of 2 types of fermented milk, represented by the prefix codes, FM and FM8.The suffix code refers to the specific replicate sample.(d) Differential metabolic pathways identified by metabolite set enrichment analysis.The color scale represents P-value.

Figure 6 .
Figure 6.Effects of adding Bifidobacterium animalis ssp.lactis Probio-M8 on the contents of organic acids, AA, and fatty acids in fermented milk.Orthogonal partial least squaresdiscriminant analysis score plots of (a) organic acids, (b) AA, and (c) fatty acids in the unfermented milk (M), milks fermented with the commercial starter culture, YoFlex Premium 1.0, with and without Bifidobacterium animalis ssp.lactis Probio-M8 (FM and FM8, respectively).(d) Heat map of organic acids, AA, and fatty acids of the 3 types of milks.The suffix code represents replicate sample.(e-g) Horizontal bar charts showing significantly different organic acids, AA, and fatty acids between FM and FM8, evaluated by Wilcoxon rank-sum test.

Figure 7 .
Figure 7. Metabolic map constructed from identified differential metabolites.Organic acids, AA, and fatty acids are written in green in the metabolic map.Upward and downward arrows represent increase and decrease in the metabolite content in probiotic fermented milk (FM8) compared with the nonprobiotic fermented milk (FM).Significant and nonsignificant changes in metabolite content are indicated by red and gray, respectively.Differential metabolites were evaluated using ANOVA at the 95% significance level (P < 0.05).CoA = coenzyme A; SCFA = short-chain fatty acid; MCFA = medium-chain fatty acid; HK pathway = hexokinase pathway; TCA = tricarboxylic acid.GLUT = glucose transporter.

Table 1 .
Sun et al.: BIFIDOBACTERIUM ANIMALIS SSP.LACTIS IN FERMENTED MILK Main nutrient contents of unfermented (± SD) Figure 1.Schematic diagram of study design.
Sun et al.: BIFIDOBACTERIUM ANIMALIS SSP.LACTIS IN FERMENTED MILK mixed with 190 μL of distilled water, filtered through 0.22-μm microporous organic membrane filters before analysis.