1. Introduction
Fermented milk is a product made from raw milk or milk powder that has been sterilized and fermented to reduce pH [
1]. Lactic acid bacteria are mainly selected for fermentation and, to a lesser extent, yeasts and molds [
2]. Compared to unfermented dairy products, they are characterized by the addition of a single probiotic or multiple probiotics for fermentation culture. The properties of different kinds of fermented milk are improved by adding appropriate amounts of ingredients, such as thickeners, functional substances, and natural plant ingredients, to improve the texture, consistency, dietary fiber content, quality features, and taste and flavor of different kinds of fermented milk. The effect of fermentation on the food matrix is manifested by the availability of a variety of nutrients and functions that enrich the fermented matrix and ultimately give the food a multi-layered taste and good organoleptic qualities in line with today’s demand for new foods [
3]. Among fermented foods, fermented milk has attracted much attention due to its great digestibility, rich taste, and ability to alleviate lactose intolerance, and accordingly, multidimensional studies have been carried out on its technical and industrial uses. Currently, most of the commercially available fermented milk is produced on the basis of cow’s milk substrate, which is more acceptable to consumers when considering the nutritional value and benefits compared to other substrates due to its safety, high nutritional value [
4], popularity in terms of taste and flavor, wide availability, as well as systematic development and promotion [
5]. More and more consumers are recognizing the benefits of fermented products, and the production and consumption of fermented milk are growing dramatically worldwide [
6].
Different kinds of fermented milk often have different fermentation characteristics, textures, and flavors, and the corresponding fermented products also show differences in composition, depending on a variety of factors in the fermentation preparation process, but the essence lies in the different fermentation characteristics of the lactic acid strains added and the differences in the final quality of the fermented milk.
S. thermophilus is more suitable for growth in the milk environment due to its long period of genetic remodeling. This, in turn, has resulted in altered carbohydrate (carbon source) utilization by
S. thermophilus in the milk environment. In particular, a strain dependence was shown for the main uptake and metabolic utilization of sugars (sucrose, glucose, galactose, fructose, etc.), with lactose being preferred.
S. thermophilus utilizes and metabolizes carbon sources through a homo-fermentation pathway, followed by glycolysis, pyruvate metabolism, and other pathways, thus generating various metabolites to complete the fermentation process. Differences in the utilization of different carbon sources by
S. thermophilus also lead to growth differences. During the fermentation process, fermented milk exhibits different fermentation characteristics at different fermentation stages, and the corresponding trends of fermentation characteristics are closely related to the final fermented products. In the fermentation system, the unique fermentation characteristics of different strains directly affect the content and metabolite composition of fermentation products. With the industrialization of fermented products and research efforts in recent years, compound strains and commercial ferments have become the mainstay of fermentation co-culture at this stage. Compound strain fermentation is more focused on a particular product characteristic or equalizing the fermentation characteristics of the fermented product, such as improving or enhancing organoleptic properties, flavor, viscosity, quality, fermentation time, etc. [
7]. While regular commercial culture focuses on the overall fermented milk product, special commercial fermenters improve the product characteristics to ensure product quality while enhancing the quality characteristics of the fermented milk, starting from the fundamental nature of the food consumed by the consumer [
8]. The high quality of food demanded by consumers and the rapid development of the fermentation industry have led to further exploration and selection of excellent fermentation strains that combine multiple fermentation advantages [
9].
S. thermophilus has become an indispensable fermenting strain for fermented dairy products due to its certified safety, strong acid production capacity, and fast fermentation speed [
10], and most of the compound strains contain
S. thermophilus in fermented products. The production and evaluation of
S. thermophilus through genomics, metabolomics, phenotypic analysis, and other research techniques are also being carried out gradually [
11]. In this paper,
S. thermophilus JM905 was isolated from traditional fermentation products; it has a good acid-producing ability, which can be used as a potential industrial fermentation strain, and the study of its fermentation and metabolic characteristics during fermentation has certain practical significance. The selection and exploitation of good fermentation strains are not limited to having certain outstanding characteristics but the overall balance of the final product quality [
12]. The screening and mining of excellent fermentation strains are not limited to having certain outstanding characteristics and the overall balance of the final product quality. A better exploration of the fermentation characteristics and metabolite fraction functions of fermented milk at different stages of fermentation and correlation with product quality [
13] would be beneficial in facilitating the preparation of fermented milk production and reducing excessive waste of resources.
Therefore, the aim of this study was to further investigate the fermentation characteristics of S. thermophilus JM905 with fermentation potential at different stages of fermentation in the milk environment and to analyze the relationship and characteristics between pH, acidity, viscosity, odor, sensory evaluation, and changes in major metabolites of the fermented milk of S. thermophilus JM905 by elucidating the utilization of various carbon source substrates. From the perspectives of substrate utilization specificity, fermentation characteristics, and metabolic components in the milk environment, we can provide theoretical data for strains with potential application in the fermentation field and provide a new direction for the selection of excellent fermentation strains.
2. Materials and Methods
2.1. Strain, Culture Medium, and Growth Conditions
S. thermophilus JM905 is isolated from a traditional fermentation product and has good fermentation potential after pre-fermentation experiments. Then, it was inoculated at 5% inoculum in an M17 broth liquid medium (Hopebio biotechnology Co., Ltd., Qingdao, China) (sterilized at 121 °C for 15 min) for activation, and the culture condition was set at a constant temperature of 37 °C for 12 h. The above procedure was repeated 2 times for the generation to ensure the maximum density of viable cells and cultured again in M17 broth liquid medium at the above proliferation rate [
14]. Fermentation was carried out with an inoculum of 10
7 CFU/mL.
2.2. Utilization of Carbon Sources by the Strains
The use of carbon sources by the strains was determined by a high-throughput microbial phenotyping system (Biolog Omnilog Technology Co., Ltd., Shanghai, China). After the strain had been incubated for 48 h by scribing, an inoculum suitable for Streptococcus was selected, and a single colony was dipped in an appropriate amount using a disposable sterile cotton swab, which was rubbed close to the top of the inoculum without touching the wall of the inoculum, and the colony tissue was sufficiently ground, inserted into the inoculum, shaken up and down slowly to avoid air bubbles, and adjusted to the appropriate turbidity [
15]. After adjusting to the appropriate turbidity, 100 μL of the inoculum with colony tissue was added to each well of the GEN III plate and incubated in the phenotyping system incubator at a temperature of 37 °C for 72 h.
2.3. Fermented Milk Sample Preparation
Skim milk powder was added to distilled water at 65 °C, fully homogenized, and sterilized at 95 °C for 20 min [
16]. The corresponding mass of powder was weighed and inoculated at 5 × 10
7 cfu/g into skim milk medium cooled below 40 °C with sucrose, and the fermentation conditions were constant temperature at 37 °C. Monitoring the pH value during fermentation, pH values of F1 (6.0 ± 0.01), F2 (5.1 ± 0.01), and F3 (4.5 ± 0.01) were selected as the indexes for the pre-, mid-, and post-fermentation stages, respectively, based on the data from the pre-fermentation experiments in the previous stage.
2.4. Physico-Chemical Analysis
2.4.1. Fermented Milk pH, Acidity, and Post-Acidification
Changes in pH during fermentation were monitored hourly by a fermentation monitor (AMS-ALLIANCE Technology Co., Ltd., Shanghai, China).
A sample of 10 mL per hour is taken and diluted in 20 mL of water to determine the pH. When the pH of the dilution is 8.3, the number of milliliters of NaOH standard solution consumed at 0.1000 moles/litre is calculated and multiplied by 10 to give the acidity of the sample, which is expressed in °T.
2.4.2. Fermented Milk Water-Holding Capacity, Viscosity, and Viable Bacteria Count
A certain amount of fermented sample mass was weighed as
m1, loaded into a centrifuge tube, and centrifuged at 5500 rpm for 30 min at low temperature; then, the supernatant was poured off, inverted for 10 min to allow the supernatant to flow out fully, and finally, the mass of the precipitate was weighed as
m2. The water-holding capacity was calculated as follows:
A total of 1 mL of each fermented milk from the pre-fermentation, middle, and post-fermentation periods (F1, F2, and F3) was taken, and serial decimal dilutions were made with 90% saline in a gradient. A total of 1 mL of fermented milk dilution was taken for each sample and incubated in the M17 agar medium incubated in the M17 agar medium of choice at 37 °C for 48 h medium of choice at 37 °C for 48 h. To determine the changes in the number of viable bacteria at different stages in fermented milk [
17].
Viscosity was measured in this test by using a DVS+ viscometer with rotor LV-4 (64) to measure the viscosity of the fermented milk at 100 rpm and taking the value at 30 s as a measure of the viscosity of the curd of the strain.
2.4.3. The Odor and Taste of Fermented Milk
Determination was carried out by an electronic nose measuring system (INSENT Technology Co., Ltd., Beijing, China). After cleaning the sensor for 60 s, a disposable sterile needle was inserted into the sample bottle as close as possible to the sample surface but without contact. The sampling interval was 1 s, the determination time was 60 s, and the determination was performed 3 times.
Determination was carried out by an electronic tongue measurement system (INSENT Technology Co., Ltd., Beijing, China). The sensor probe should be soaked in a buffer solution for 24 h and cleaned. The sample is homogenized using a homogenizer, extracted by magnetic stirring for 20 min, mixed thoroughly, and centrifuged at 6500 rpm for 10 min by freezing. Then, the supernatant is extracted using a double layer of medium-speed quantitative filter paper, and the filtrate is diluted to 100 mL and poured into a special beaker for the electronic tongue to be measured 3 times.
2.5. Determination of Fermented Milk Protein and Free Amino Acids
The protein content of fermented milk was determined using an automated Kjeldahl nitrogen tester (HITACHI Technology Co., Ltd., Shanghai, China), and all experiments were repeated 3 times in parallel.
After acid digestion, fermented milk was determined using an amino acid analyzer.
2.6. Determination of Fermented Milk Fat and Fatty Acids
A quantity of fermented milk sample was taken and diluted using sterile PBS solution, and the fat in it was extracted by hydrolysis ether solution. After hydrolysis, the fat was concentrated to dryness using a rotary evaporator, and the residue was the fat extract, which was subjected to saponification and methyl esterification of fatty acids directly after the addition of the internal standard [
18].
The fermented milk was subjected to dilution treatment, and the alkaline (ammonia) hydrolysate of the sample was extracted with anhydrous ether and petroleum ether; the solvent was removed by distillation or evaporation, and the content of the extracted fat dissolved in the solvent was determined [
19].
2.7. Sensory Evaluation
Sensory evaluation was carried out by 30 sensory assessors in the sensory evaluation room of the Northeast Agricultural University. (15 males and 15 females, 22–27 years of age, none with a clear taste preference). All samples were placed undifferentiated and numbered randomly, and pure water was provided for mouth rinsing between tasting different numbered samples. The following assessment criteria were established: freshness, acidity, sweetness, odor, texture, and acceptability. The sensory assessors were asked to evaluate the taste by using a 9-point scale (9, first sensation on the palate and persistent; 8, accompanied throughout the senses; 7, pleasant taste present; 6, pleasant taste present but not persistent; 5, normal taste and persistent; 4, unpleasant sensation but brief; 3, unpleasant sensation present; 2, unpleasant sensation and persistent; 1, very unpleasant sensation present) assessed after overall sensory scoring.
2.8. Metabolic Composition of Fermented Milk
Pipette 100 μL of each of the pre-, mid-, and post-fermentation samples into an EP tube, add 400 μL of extract (methanol: acetonitrile = 1:1 (V/V), containing isotopically labeled internal standard mixture), vortex and mix for 30 s; sonicate for 10 min (ice water bath); leave at −40 °C for 1 h. Centrifuge samples at 4 °C, 12,000 rpm (centrifugal force 13,800× g, radius 8.6 cm). The samples were centrifuged for 15 min; the supernatant was removed from the injection vial and tested on the machine; and all samples were mixed with an equal amount of supernatant to form QC samples for detection by ultra-high performance liquid chromatography (UHPLC). UHPLC (BIOTREE Biotechnology Co., Ltd., Shanghai, China) chromatographic conditions: column temperature 25 °C; flow rate 0.5 mL/min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia; B: acetonitrile; gradient elution program: 0–0.5 min, 95% B; 0.5–7 min, B from Q–TOF mass spectrometry conditions: ESI source setup parameters: sheath gas flow rate of 50 Arb, Aux gas flow rate of 15 Arb, capillary temperature of 350 °C. Arb, capillary temperature 350 °C, full MS resolution 60,000, MS/MS resolution 30,000, collision energy 20/30/40 in NCE mode, injection voltage 3 kV (positive) or −3 kV (negative).
For non-targeted metabolomics studies, the positive and negative ion modes were selected for full analysis, and the positive and negative ion data were integrated for analysis.
2.9. Data Analysis
Each experiment was repeated three times independently, and the results are expressed as mean Earth standard deviation (SD). SPSS software (SPSS 18.0, Inc, Chicago, IL, USA) was used to analyze the experimental data. p-values < 0.05 were statistically significant. Plots of the experimental data were generated using Origin software (Origin 2018, Inc., Chicago, IL, USA). The non-target metabolism data were analyzed by BIOTREE Biotechnology Co., Ltd., Shanghai, China.
4. Discussion
In recent years, the rise of fermented dairy products has been fueled by a significant increase in consumer attention to food nutrition and safety, prompting a further shift towards higher quality and functionality. It is required not only to expand its application in actual production but also to fully investigate its characteristics during the fermentation process of the corresponding substrates, which needs to be verified by continuous practical operation. Following this, the fermentation characteristics and special metabolites that have been identified are used to target and enhance the nutrients in fermented milk or to conduct other functional studies. The fermentation index is the most intuitive response to the fermentation performance of fermentation strains and the final fermented milk physicochemical state, and the results ultimately determine whether the strain has a strain that can be applied to the actual production. Unsuitable strains or strains with certain fermentation defects may show unsatisfactory results in one of the physicochemical indexes, such as long fermentation time, severe post-acidification of the final fermented milk, or poor curd condition. The key to determining the fermentation rate of a strain is the genetic differences of the strain. Studies have shown that lactate dehydrogenase (LDH) is a key enzyme in the metabolic pathway of lactic acid production by Lactobacillus, which can catalyze the conversion of pyruvic acid into lactic acid. According to the classification of the catalytic products produced, it can be classified into D-lactate dehydrogenase and L-lactate dehydrogenase, which can produce D-lactic acid and L-lactic acid, respectively, whereas the overall tendency of L-lactate is to increase continuously, thus reducing the pH and promoting the fermentation [
22]. The fermented milk components and the type of metabolites determine the quality and nutrition of fermented milk. To resolve the deficiency of fermentation strains, it is necessary to supplement the deficiency of single-strain fermentation by compounding more strains, so it is extremely important to explore excellent fermentation strains in the field of fermentation.
Key metabolite components in fermented milk have a significant impact on the quality and fermentation characteristics of fermented dairy products [
23]. Non-targeted sequencing technologies allow for a more specific understanding of the composition of individual substances by qualitatively annotating biological information in sample components [
24]. Based on the annotated substance composition, specific enhancement or extraction of beneficial components or classes in fermented milk can be performed to improve the quality of different kinds of fermented milk or to conduct corresponding functional studies. Thus, the study of metabolite fractions and trends in fermented milk contributes to a more complete understanding of the quality and fermentation characteristics of a particular strain. The results of the fermentation characterization showed that the pH value of Streptococcus thermophilus JM905 decreased slowly in the early stage of fermentation and changed more significantly in the later stage. A titratable acidity of 87 °T was reached at the completion of fermentation, and
S. thermophilus JM905 had better acid production characteristics than the same strain fermenting milk [
25]. For metabolic annotation to organic acids, benzoic acid, 2-hydroxybutyric acid, folic acid, L-lactic acid, and methylglutaric acid content also increased, which is the same trend as the overall fermentation acid production. The folic acid content continued to decrease, and related studies showed that except for Lactobacillus plantarum, which showed an increase in folic acid content after fermentation, the rest of the strains showed a bottom in folic acid content, which was related to the strain species specificity [
26]. In contrast, the results of this paper confirm that the folate content continued to decrease throughout the fermentation process, presumably due to the involvement of other substance production pathways. The key indicator for determining whether fermented milk has reached the fermentation endpoint is pH, which is judged by the amount of acid produced by the strain in the substrate. The results showed a gradual decrease in pH and a gradual increase in acidity with the duration of fermentation, with a more pronounced change in the later stages of fermentation. In an acidic environment with decreasing pH, the degree of protein denaturation gradually increases, and the water retention capacity of
S. thermophilus JM905 fermented milk increases, which is consistent with the fermentation results and the trend of the above study [
27]. The degree of denaturation of proteins in fermented milk, the aggregation of casein in an acidic environment, and the type and content of extracellular polysaccharides are related to the water-holding capacity and viscosity of fermented milk. Increased acid production by
S. thermophilus JM905 in the fermentation environment leads to protein denaturation in milk. The water-holding capacity of fermented milk is due to the exposure of hydrophobic groups when the proteins in milk are denatured by acid, resulting in reduced diffusivity, intermolecular collisions, and aggregation, leading to an increase in viscosity, and a continuous upward trend in overall viscosity values [
28]. Extracellular polysaccharides in fermented milk confer fermented milk viscosity due to the tight junctions and disordered arrangement of monosaccharides. Annotation to the sugar alcohols such as D-glycero-D-galacto-heptitol, D-Arabinose, Sedoheptulose, and others showed a continuous increase in the content of polysaccharides, of which galactose and D-Arabinose were the main constituents constituting the polysaccharides, increasing the degree of viscosity, which is similar to the metabolic annotation results of the present study [
29]. The polysaccharide components in improving the texture state in the fermented milk sensory was more obvious in the first and middle stages, which made the milk state appear viscous and pulling, and in the later stage, the texture state appeared to be coagulated and blocky, and it was not possible to discern the effect of its influence.
As for the carbon source utilized by the strain, the results of this paper showed a high utilization of the carbon source D-galactose, which is usually present in the milk matrix as a structural part of lactose, and based on the study of the carbon source of
Streptococcus thermophilus, it can be hypothesized that D-galactose is under-utilized during fermentation due to the addition of an additional carbon source [
30]. The degree of utilization of carbon sources such as sucrose, fructose, mannose, glucose, and galactose by Streptococcus thermophilus was related to the corresponding utilization of the uptake system, as evidenced by the phenotypic traits, which is in agreement with the results of carbon source utilization in this paper [
31]. The efficiency of carbon source utilization by Streptococcus thermophilus, as the main strain of fermentation, directly affects its fermentation characteristics and the metabolites presented by its products, regardless of the substrate in which it is fermented and the efficiency or sequence of good utilization of a variety of carbon sources affects the fermentation performance to a certain extent.
The composition of the main substances in fermented milk affects the presentation of flavors. Detecting changes in food odor with biosensors can show the production and trends of different substances and can also be used as a method to detect spoilage of food components (including fats, carbohydrates, and proteins) that cannot be easily removed by the human senses [
32]. Organic acids are an important source of odor for fermented lactic acids (e.g., lactic and oxalic acid) [
33]. The improvement of taste and odor by
S. thermophilus JM905 was gradual and continuous. Sourness and alkanes had the most evident correlation with the pH of the fermented milk. The study also showed that as fermented products, the degree of alteration of sourness was primary, followed by freshness [
34], which had a positive correlation with glutamic acid and aspartic acid. The intensity of freshness, which is involved in compositional richness, had a lower correlation with the above-mentioned components. It was also found that alkanes, sulfury compounds, and alcohol compounds showed some negative correlation with savory flavor, richness, and freshness. The results of some studies have shown that the production of fatty acids and several specific amino acids are also the main components contributing to changes in acidity. There is a strong correlation between fatty acids and fat content [
35]. The results of this experiment showed that various types of fermented milk were low in fat, monounsaturated, and polyunsaturated fatty acids. It has been shown that the fermentation substrate affects the fatty acid content of fermented milk. In this experiment, skimmed milk was chosen as the substrate, which resulted in a lower fat content after fermentation, which in turn affected the fatty acid content. As for the effect of small molecules in fermented milk on sensory evaluation, the addition of free amino acids produced by the fermentation of lactic acid bacteria to low-fat dairy products improves organoleptic palatability after consumption and imparts a creamy flavor and satiety. Different amino acids have different flavors, with aspartic acid and glutamic acid imparting freshness and acidity to fermented milk. Sweet amino acids are less hydrophobic, while bitter amino acids are more hydrophobic, and the corresponding flavor amino acid content affects the flavor of different kinds of fermented milk to different degrees, which together constitute the multi-layered flavor of fermented milk.
Various components are produced by strains during the fermentation process, and the addition of different components can have relevant effects on the fermentation characteristics, metabolites, and potential quality functions of the final fermented milk. This paper focuses more on analyzing the beneficial metabolites, such as polysaccharides and amino acids, that affect the quality and metabolic properties of fermented milk and does not conduct in vitro tests on specific components of fermented milk corresponding to a certain functionality, but functionality tests of fermented milk have also become a hot research topic.
In conclusion,
S. thermophilus JM905 can enrich milk with a series of nutrients and characteristic metabolites related to the quality and fermentation properties of fermented milk. Research on the metabolites of different kinds of fermented milk and their fermentation characteristics continues, with beneficial metabolites providing additional quality advantages to fermented milk. The addition of beneficial fermentation strains to the original fermented milk also positively affects the content of beneficial metabolites [
36]. This lays the foundation for subsequent studies on the corresponding functions and targeted improvement of the quality of fermented products, as well as the quality control of different kinds of fermented milk.