Preliminary Study on the Application of Protease-Producing Lactiplantibacillus plantarum in Yogurt Fermentation

Abstract

Starter culture significantly influences the texture and flavor of yogurt, making the selection of appropriate fermentation strains a key focus in yogurt starter research. In this study, protease-producing Lactiplantibacillus plantarum NH-24, identified in prior experiments, was combined with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophiles for yogurt fermentation. Indicators such as coagulation state, acidity, and water-holding capacity were measured to determine the optimal fermentation temperature and starter ratio. Additionally, the effects of this strain on the yogurt’s texture, sensory properties, and volatile flavor compounds were evaluated. The results indicate that a fermentation temperature of 37 °C and a starter ratio of 4:4:3 were most suitable for yogurt production. Further analysis demonstrated that incorporating Lp. plantarum NH-24 improved the yogurt’s texture and flavor while reducing post-acidification during storage. Thus, protease-producing Lp. plantarum NH-24 holds significant promise as a yogurt starter culture.

1. Introduction

Lactic acid bacteria are among the essential microorganisms used in the production of fermented foods, and the screening and isolation of protease-producing lactic acid bacteria have consistently been a focal point in strain selection research. During fermentation, the extracellular proteases produced by lactic acid bacteria can enhance the flavor and quality of food products.

In the food industry, lactic acid bacteria and their metabolites play a crucial role in breaking down raw materials during fermentation, releasing or synthesizing bioactive components that are beneficial to human health. This process not only improves the sensory qualities and nutritional properties of food but also effectively extends its shelf life [1]. The fermentation process involving lactic acid bacteria significantly contributes to the development of food flavors. For example, Lactiplantibacillus plantarum has been shown to significantly alter the levels of flavor compounds such as ethyl acetate, acetic acid, and methyl isobutyrate during rice wine fermentation [2]. Lactiplantibacillus plantarum LCC-605 secreted spherical exopolysaccharide nanoparticles (EPS-605 NPs), which may contribute to the quality, function, and stability of the fermented yogurt, significantly improved the water holding capacity (71.7 ± 0.5 %), and the texture of the yogurt [3]. The application of the high-selenium-tolerant strain Lp. plantarum NML21 in the biosynthesis of selenium-enriched yogurt has markedly augmented the ketonic flavor compounds within the product, with notable elevations in the concentrations of 2-heptanone and 2,3-pentanedione [4]. Furthermore, Lp. plantarum 423 enhances the concentration of flavor substances like sulfides and aromatic hydrocarbons during the fermentation of rice bran and wheat bran, while simultaneously boosting the antioxidant capacity of the fermentation broth [5].

As microorganisms extensively utilized in food fermentation, lactic acid bacteria have attracted considerable attention owing to their proteolytic activity [6]. In microbial fermentation systems, proteolytic enzymes produced by Lactobacillus and Lactococcus species exhibit significant catalytic effects on the degradation of casein, leading to the formation of bioactive peptides. This process markedly improves the flavor and texture of fermented dairy products [7].

Yogurt is a widely popular fermented dairy product, favored by consumers for its excellent flavor, appealing texture, and high nutritional value [8]. It is rich in proteins, carbohydrates, vitamins, and other health-beneficial components [9], contributing to its extensive market demand. Most yogurt is produced through co-fermentation using Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus as starter cultures [10]. The co-fermentation process involving Lactobacillus species can significantly improve the flavor, quality, and storage stability of fermented milk products [11,12].

The texture, mouthfeel, and flavor of yogurt are key factors that determine consumer acceptance [13]. As a result, when introducing selected lactic acid bacteria strains for co-fermentation, it is essential to evaluate multiple factors to ensure their effectiveness and suitability as commercial starter cultures for use in the industrial-scale production of food products.

In the present investigation, a protease-producing strain of Lactiplantibacillus plantarum NH-24, isolated through systematic screening, was employed as a microbial starter in yogurt fermentation. By analyzing the properties of the mixed yogurt, the impact of Lp. plantarum NH-24 on the texture, mouthfeel, flavor, and other attributes of yogurt were explored. The findings revealed that yogurt containing Lp. plantarum NH-24 demonstrated ideal coagulation time, superior coagulation quality, balanced acidity, and optimal water-holding capacity. Additionally, it received the highest sensory evaluation ratings, improved yogurt texture, enhanced taste, and prolonged shelf life by reducing post-acidification.

2. Materials and Methods

2.1. Revitalization of Microbial Strains for Experiments

The Lactobacillus delbrueckii subsp. bulgaricus (BK14E2) and Streptococcus salivarius subsp. thermophilus (CK09B2) strains used in this study were obtained from Junyao Runying Biotechnology (Shanghai) Co., Ltd. (Shanghai, China). Additionally, Lactiplantibacillus plantarum N (unpublished data) was also assessed as a safe probiotic.

The cryopreserved L. bulgaricus and S. thermophilus were first cultured on MRS agar at 37 °C for 48 h, followed by inoculation into MRS broth and further incubation at 37 °C for 24 h.

Lp. plantarum NH-24, isolated from cheese (Tacheng, Xinjiang), demonstrates high protease production. The strain was grown on MRS agar at 37 °C for 48 h and then transferred to MRS broth for further incubation at 37 °C for 24 h.

2.2. Experimental Materials

The fresh milk (Bright Dairy “You Be”) used for yogurt preparation was purchased from Lawson (Shanghai, China), and the maltose powder was supplied by Shengfa Biotechnology (Zhengzhou, China).

2.3. Optimization of Mixed Yogurt Formulation Ratios

The activated Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, and Lactiplantibacillus plantarum NH-24 were used to ferment yogurt at a 1.5% inoculation rate in ratios of 4:4:1 (Group A), 4:4:3 (Group B), and 4:4:5 (Group C) [14]. Controls included yogurt fermented with L. bulgaricus and S. thermophilus (4:4, Group D) and yogurt fermented solely with Lp. plantarum NH-24 (Group E). Fermentation was performed at 37 °C and 42 °C (labeled as A-1, A-2, B-1, B-2, etc.). The optimal temperature and ratio were selected based on coagulation state, coagulation time, acidity, water-holding capacity, viable counts, and sensory evaluation.

2.3.1. Preparation of Mixed Yogurt

The fresh milk was preheated to 50 °C, blended with maltose powder, and sterilized at 95 °C for 5 min. After cooling to room temperature, the three strains were inoculated into the sterilized milk at the specified ratios (add 1.5% (w/v) starter culture and 6% (w/v) maltose powder to 100 mL of pure milk), followed by fermentation at 37 °C and 42 °C (Streptococcus (S.) thermophilus and Lactobacillus (L.) delbrueckii ssp. bulgaricus are widely used as a combined starter culture for milk fermentation, often at temperatures of 37 °C and 42 °C) [15], the fermentation period is determined by the time necessary for the milk to achieve complete coagulation.

2.3.2. Determination of Coagulation State and Time

The yogurt was prepared as described in Section 2.3.1, with the coagulation state and time (fermentation endpoint) monitored and recorded for each group.

2.3.3. Determination of Acidity During Fermentation and at the Fermentation Endpoint

The preparation process of yogurt was as described in Section 2.3.1. The acidity of the yogurt was measured during fermentation over a time range of 0 to 12 h, with measurements taken every three hours. The acidity determination was performed in accordance with the National Food Safety Standard: Determination of food acidity (GB 5009.239-2016 [16]).

Determination of pH value: The pH value was measured using a pH meter (Shanghai Xinsen Instrument Factory, Shanghai, China) once the sample had cooled to room temperature.

Titration acidity determination [17]: Add 20 mL of freshly boiled and cooled water to a 150 mL conical flask. Weigh 10 g of the homogenized sample and add it to the flask, followed by 2 mL of phenolphthalein indicator solution (Macklin Biochemical Technology Co., Ltd., Shanghai, China). Mix well. Titrate with a 0.1000 mol/L sodium hydroxide standard solution (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) until a faint pink color develops and remains stable for 30 s. Record the volume (in mL) of sodium hydroxide solution consumed and calculate the titration acidity using the provided formula.

Acidity (°T) = (C × V × 100)/(M × 0.1)

C: the molar concentration of the NaOH standard solution (mol/L), V: the volume of the NaOH standard solution consumed in titration (mL), M: the mass of the sample (g).

The result is expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, rounded to three significant figures.

2.3.4. Determination of Water-Holding Capacity at Fermentation Endpoint

Water retention measurement [18]: Weigh an empty centrifuge tube. Add 10 g of sample to the tube and record the total weight. Centrifuge at 6000 r/min for 20 min at 4 °C, allow to stand for 10 min, then discard the supernatant. Measure the remaining mass. Perform the procedure in triplicate and calculate water retention using the provided formula.

Water retention (%) = (W3 − W1)/(W2 − W1) × 100%

W1: the mass of the empty centrifuge tube (g), W2: the total mass of the centrifuge tube with the sample (g), W3: the remaining mass of the centrifuge tube (g).

2.3.5. Determination of Lactic Acid Bacteria Count at the End of Fermentation

Lactic acid bacteria count determination: Refer to the National Food Safety Standard: Microbiological Examination of Food—Examination of Lactic Acid Bacteria (GB 4789.35-2023 [19]).

2.3.6. Detection of Coliforms, Molds, and Yeast

Sample preparation: Performed following National Food Safety Standard: General Principles for Microbiological Testing of Food (GB 4789.1-2016 [20]) and National Food Safety Standard: Microbiological Testing of Food—Sampling and Sample Preparation for Milk and Dairy Products (GB 4789.18-2024 [21]).

Coliform analysis: Refer to National Food Safety Standard: Microbiological Testing of Food—Coliform Count (GB 4789.3-2016 [22]).

Mold and yeast analysis: Refer to National Food Safety Standard: Microbiological Testing of Food—Mold and Yeast Count (GB 4789.3-2016).

2.3.7. Sensory Evaluation of Mixed Yogurt

Sensory evaluation was conducted following the National Food Safety Standard: Fermented Milk (GB 19302-2010 [23]) with adjustments. A panel of 20 evaluators (10 male, 10 female) assessed the yogurt in a well-equipped laboratory. Attributes evaluated included color (20 points), aroma (20 points), texture (30 points), and consistency (30 points), totaling 100 points (

Table 1. Sensory evaluation criteria for yogurt.

Test items	Characteristics	Evaluation Criteria
color (20 points)	Uniform color, bright luster, and creamy white curd	15~20
	Uniform color, glossy, creamy white or light-yellow curd	10~14
	Locally uneven in color, slight luster, and pale-yellow curd	5~9
	Uneven color, lacking luster, brown or abnormal-colored curd	<5
aroma (20 points)	Distinct yogurt aroma, rich dairy fragrance, no off odors	15~20
	Moderate yogurt aroma, moderately rich dairy fragrance, no off odors	10~14
	Faint yogurt aroma, weak dairy fragrance, no pronounced off odors	5~9
	No yogurt aroma, no dairy scent, with off odors	<5
texture (30 points)	Smooth and tender texture, no graininess, balanced sweetness and acidity	20~30
	Smooth and tender texture, no noticeable graininess, moderately balanced sweetness and acidity	10~19
	Slightly coarse, with a hint of graininess, either overly sour or overly sweet	5~9
	Coarse texture, strong graininess, either excessively sour or excessively sweet	<5
consistency (30 points)	Smooth surface, fine and uniform texture, no whey separation, no bubbles	20~30
	Smooth surface, uniform texture, slight whey separation, few bubbles	10~19
	Rough surface, uneven texture, curd formation present, slight layering, partial whey separation, and presence of bubbles	5~9
	Rough surface, coarse texture, significant curd formation, severe layering, extensive whey separation, and numerous bubbles	<5

) [24]. Each sample was tasted three times, with purified water used for rinsing between tastings.

2.4. Research on the Fermentation Characteristics of Mixed Yogurt

Using the results from Section 2.3, the optimal ratio of the three strains, incubation temperature, and coagulation time for the mixed yogurt were determined. Its fermentation properties were evaluated, including texture, volatile flavor compounds, electronic nose and tongue analyses, and acidity changes during storage.

2.4.1. Determination of Textural Properties of Mixed Yogurt

Using a TA-XT Plus texture analyzer (Stable Micro Systems, Godalming, UK) in TPA mode, yogurt texture properties such as hardness, springiness, cohesiveness, chewiness, and resilience were evaluated [25]. Parameters included a P6 cylindrical probe (28.27 mm² contact area), Auto-10 g trigger, 20 mm test distance, 75% compression, and speeds of 6 mm/s (pretest), 2 mm/s (test), and 2 mm/s (post-test).

2.4.2. Analysis of Volatile Flavor Compounds in Mixed Yogurt

Volatile flavor compounds in the blended yogurt were analyzed using Gas Chromatography–Ion Mobility Spectroscopy (GC-IMS, Dortmund G.A.S., Dortmund, Germany). Samples, carried by gas, were first separated in the GC column and then ionized in the mobility tube. Migration to the Faraday disk facilitated secondary separation, enabling qualitative analysis.

Headspace Conditions [26]: Place 2 g of sample in a 20 mL vial, incubate at 60 °C for 15 min, and inject 500 μL. Injection needle: 65 °C; agitation: 500 r/min. GC Setup: Column: MXT-WAX capillary; temperature: 60 °C; runtime: 20 min; carrier gas: high-purity nitrogen. Pressure program: 2.0 mL/min (2 min), ramp to 10.0 mL/min (8 min), then to 100.0 mL/min (10 min), and hold for 40 min. IMS Parameters: Ionization: tritium source (³H); tube length: 53 mm; field strength: 500 V/cm; temperature: 45 °C; drift gas: high-purity nitrogen; flow rate: 75.0 mL/min; mode: positive ion.

2.4.3. Electronic Nose Analysis of Mixed Yogurt

Yogurt aroma was analyzed using a Fox4000 electronic nose (Alpha MOS, Toulouse, France). Samples were homogenized, placed in 10 mL headspace vials, and incubated at 50 °C for 30 min [27]. Detection lasted 120 s with 1 s intervals, sensor cleaning for 120 s, and injection at 500 μL/s [28]. Sensor types are detailed in

Table 2. Electronic nose sensors.

Number	Sensor Type	Sensed Material Type
1	LY2/LG	Fluorine and nitrogen oxides
2	LY2/G	Ammonia, amines, carbon compounds
3	LY2/AA	Ethanol, oxygen-containing compounds
4	LY2/GH	Alcohol, acetone, ammonia
5	LY2/gCTL	Hydrogen sulfide, ammonia, amine compounds
6	LY2/gCT	Carbon monoxide, methane, and other hydrocarbons
7	T30/1	Organic compounds, polar compounds, hydrogen chloride
8	P10/1	Hydrocarbon
9	P10/2	Nonpolar compounds
10	P40/1	Fluorine
11	T70/2	Methylbenzene, xylene
12	PA/2	Ethanol, ammonia, amine compounds
13	P30/1	Hydrocarbon, combustion products
14	P40/2	Fluorides and sulfides
15	P30/2	Hydrogen sulfide, ketone
16	T40/2	Chlorine
17	T40/1	Fluorine
18	TA/2	Ethanol

.

2.4.4. Electronic Tongue Analysis of Mixed Yogurt

Flavor analysis was performed using an ASTREE II LS16 electronic tongue (Alpha MOS, Toulouse, France) [29]. A 50 mL yogurt sample was diluted with 150 mL distilled water, homogenized, and centrifuged at 6000 r/min for 15 min (LYNX 4000, Thermo Fisher, Waltham, MA, USA). The filtered supernatant was analyzed. The system includes seven taste sensors, with sensitivities detailed in

Table 3. Electronic tongue sensors.

Number	Sensor Categories	Sensitive Taste Information
1	AHS	Sourness
2	PKS	General purpose
3	CTS	Saltiness
4	NMS	Umami
5	CPS	General purpose
6	ANS	Sweetness
7	SCS	Bitterness

.

2.4.5. Determination of Acidity in Mixed Yogurt During Storage

Yogurt acidity changes during storage were measured every 5 days, with triplicate testing each time, following the method outlined in Section 2.3.3.

2.5. Statistical Analysis

The graphs were plotted using Origin 2018, and data analysis was conducted using SPSS 23.0. Significant differences were evaluated through analysis of variance (ANOVA) followed by Duncan’s multiple comparison test. All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation. p < 0.05 was considered statistically significant.

3. Results

3.1. Screening Results of the Proportion of Mixed Yogurt

3.1.1. Screening Results of Curd State and Time

The coagulation characteristics and time of yogurt samples were evaluated at two different temperatures, 37 °C and 42 °C, with varying concentrations of Lactiplantibacillus plantarum NH-24 (groups A, B, C) and two control groups (D, E). Comparative analysis revealed that the coagulation quality of all samples was consistently better at 37 °C than at 42 °C (At 42 °C, whey separation occurs in the yogurt, accompanied by a weakened and loosely structured coagulum). Consequently, the 37 °C fermentation condition was selected for a detailed evaluation of coagulation properties.

By observing the curd state (Figure 1), it was noted that groups A and C exhibited a slight yellow color, while groups B, D, and E all presented a milky white appearance. Group A exhibited minor defects, including bubble formation and whey separation. In contrast, groups B, D, and E demonstrated optimal coagulation properties, characterized by bubble-free, dense structures. Group C showed intermediate characteristics with noticeable whey separation and a coarser texture.

Based on these observations, it was concluded that 37 °C represents the optimal fermentation temperature, with groups B, D, and E demonstrating superior coagulation characteristics under these conditions.

By comparing the coagulation times (

Table 4. Coagulation times.

Sample	Coagulation Times (h)	Sample	Coagulation Times (h)
A-1 (1)	10.32 ± 0.19 b(2,3)	A-2	9.03 ± 0.21 b
B-1	10.04 ± 0.08 bc	B-2	8.61 ± 0.13 b
C-1	9.86 ± 0.10 c	C-2	8.61 ± 0.27 b
D-1	8.64 ± 0.13 d	D-2	7.58 ± 0.14 c
E-1	18.79 ± 0.32 a	E-2	23.08 ± 0.38 a

⁽¹⁾ A-1 (fermented at 37 °C, blending ratio of 4:4:1); B-1 (fermented at 37 °C, blending ratio of 4:4:3); C-1 (fermented at 37 °C, blending ratio of 4:4:5); D-1 (fermented at 37 °C, blending ratio of 4:4, without Lp. plantarum NH-24); E-1 (fermented at 37 °C, pure Lp. plantarum NH-24 fermentation); A-2 (fermented at 42 °C, blending ratio of 4:4:1); B-2 (fermented at 42 °C, blending ratio of 4:4:3); C-2 (fermented at 42 °C, blending ratio of 4:4:5); D-2 (fermented at 42 °C, blending ratio of 4:4, without Lp. plantarum NH-24); E-2 (fermented at 42 °C, pure Lp. plantarum NH-24 fermentation); ⁽²⁾ The results present the mean ± standard deviation (n = 3); ⁽³⁾ Lowercase letters (a, b, c, etc.) are used to indicate significant differences, with different letters within the same column representing statistically significant differences (p < 0.05), while the same letters denote no significant difference.

), it was observed that the coagulation time was shorter when 42 °C was used as the fermentation temperature. The yogurt in group D had the shortest coagulation time, while the coagulation time increased with the higher proportion of Lp. plantarum NH-24 added, with group E exhibiting the longest coagulation time. The extended coagulation duration noted in Group E is likely due to the comparatively diminished acidogenic potential of Lp. plantarum NH-24. The elongation of the coagulation period could further be ascribed to the fact that a temperature of 42 °C is not conducive to the optimal growth of Lp. plantarum NH-24, thereby diminishing its rate of proliferation and subsequently influencing the yogurt fermentation dynamics. This indicates that blending the starter cultures can effectively reduce the coagulation time.

3.1.2. Acidity Measurement Results During Fermentation and at the Endpoint of Fermentation

The pH of yogurt consistently decreased throughout the fermentation process (Figure 2). At 37 °C (Figure 2A), the pH decline rate of the composite yogurt varied, starting slowly, accelerating between 6 and 9 h, and then slowing again. Group E-1 showed the smallest pH reduction, followed by A-1, while the other three groups exhibited similar trends. After 12 h, all groups except E-1 reached a pH of around 4.5, whereas E-1 only decreased to approximately 5.0. At 42 °C (Figure 2B), the pH declined rapidly during the 0–3 h and 6–9 h intervals. Group E-2 experienced the least pH reduction, stabilizing at around 5.0 after 12 h, while the other groups reached approximately 4.5 by 9 h.

Acid production capacity is a key criterion for evaluating the suitability of microbial strains as starter cultures, as it directly impacts the sensory attributes and flavor profile of fermented products [30]. Acidity levels, which indicate this capacity, are regulated by national standards, mandating a minimum acidity of 70 °T for yogurt. However, excessive acidity can impair lactic acid bacteria viability, promote milk protein denaturation, and negatively affect product texture. Furthermore, it may compromise digestibility, nutrient absorption, and gut health [31].

The acidity of all yogurt groups increased progressively with fermentation time (Figure 3). Yogurt fermented at 42 °C consistently exhibited higher acidity levels compared with those at 37 °C within equivalent time intervals. Under 37 °C fermentation (Figure 3A), the rate of acidity increase accelerated during the first 9 h but slowed between 9 and 12 h. Group E-1 demonstrated the lowest acidity, and by the 12 h mark, all groups except E-1 had achieved an acidity of 70 °T. The acidity ranking across the groups was D-2 > B-2 > C-2 > A-2 > E-2. At 42 °C (Figure 3B), all groups except E-2 surpassed 70 °T by approximately 9 h.

The acidity of yogurt groups A, B, C, and D was higher and the pH lower when fermented at 42 °C, while group E showed the opposite trend (Figure 4). The composite starter culture demonstrated enhanced acid production capabilities compared with fermentation solely with Lp. plantarum NH-24, making it more suitable for yogurt production (Figure 4B).

3.1.3. Results of the Determination of Water-Holding Capacity and Lactic Acid Bacteria Count

The water-holding capacity of yogurt refers to the ability of its protein gel network to retain water [32], playing a crucial role in maintaining the texture and stability of yogurt [33]. A reduction in the pore size of the internal protein gel network enhances the restricted permeability and water-holding capacity of the casein gel, facilitating the formation of a tightly structured set yogurt [34].

The yogurt in Groups A, B, C, and D demonstrated higher water-holding capacities when fermented at 42 °C, whereas Group E displayed the opposite trend (Figure 5). Among all the groups, Group B, with a bacterial strain ratio of 4:4:3, exhibited the highest water-holding capacity.

As stipulated by the national standard GB 19302-2010, the lactic acid bacteria count in fermented milk must be no less than 1 × 10⁶ CFU/mL. Measurements of the lactic acid bacteria counts in yogurt samples from each group revealed that all groups surpassed this threshold, complying with the fermented milk standard (

Table 5. Lactic acid bacteria count.

Sample	Lactobacillus Count (×108 CFU/(mL))	Streptococcus thermophilus Count (×108 CFU/(mL))	Lactic Acid Bacteria Count (×108 CFU/(mL))
A-1 (1)	8.67 ± 0.06 c(2,3)	8.33 ± 0.06 e	17.00 ± 0.10 c
B-1	9.70 ± 0.10 b	9.17 ± 0.12 c	18.87 ± 0.15 b
C-1	8.43 ± 0.15 c	8.93 ± 0.06 d	17.37 ± 0.12 c
D-1	7.63 ± 0.06 d	6.43 ± 0.15 f	14.07 ± 0.15 d
E-1	2.67 ± 0.06 e	1.83 ± 0.06 g	4.50 ± 0.10 e
A-2	9.73 ± 0.06 b	9.17 ± 0.12 c	18.90 ± 0.06 b
B-2	10.67 ± 0.58 a	9.83 ± 0.06 a	20.50 ± 0.61 a
C-2	10.33 ± 0.58 a	9.63 ± 0.12 b	19.97 ± 0.64 a
D-2	8.90 ± 0.10 c	8.30 ± 0.10 e	17.53 ± 0.68 c
E-2	1.87 ± 0.15 f	1.27 ± 0.06 h	3.13 ± 0.21 f

⁽¹⁾ As presented in Table 4. ⁽²⁾ The results present the mean ± standard deviation (n = 3). ⁽³⁾ Lowercase letters (a, b, c, etc.) are used to indicate significant differences, with different letters within the same column representing statistically significant differences (p < 0.05), while the same letters denote no significant difference.

). A comparison of the data across groups indicated that yogurt fermented at 42 °C contained a higher lactic acid bacteria count than that fermented at 37 °C. Specifically, Group B recorded the highest lactic acid bacteria count, whereas Group E had the lowest. Consequently, the compound starter culture proves more suitable for yogurt fermentation compared with using Lp. plantarum NH-24 alone.

3.1.4. Results of Microbial Limit Testing

According to the microbial limit regulations outlined in GB 19302-2010, the compound yogurt was tested for coliforms, molds, and yeasts. None of these microorganisms were detected in any of the yogurt groups, confirming compliance with the national standard for microbial limits.

3.1.5. Results of Sensory Evaluation

The sensory evaluation results of each yogurt group were statistically analyzed (Figure 6). Yogurt fermented at 37 °C scored higher in color, flavor, texture, consistency, and overall score compared with that fermented at 42 °C. Among the yogurt groups fermented at 37 °C, Group B-1 achieved the highest scores across all criteria, indicating the best sensory outcomes. Yogurt from Group B-1 exhibited a uniformly bright, milky-white curd, a pronounced characteristic yogurt aroma, a smooth texture without graininess, a glossy surface, a fine and consistent structure, no bubble formation, and no whey separation. Group B-1 demonstrated the best sensory results.

3.2. Research on the Fermentation Characteristics of Mixed Yogurt

Based on the measurement results from Section 3.1, it can be comprehensively inferred that Group B-1 exhibited an appropriate coagulation time, excellent curd formation, moderate acidity, and the best water-holding capacity. All its indicators complied with national standards, and it achieved the best sensory evaluation results. Therefore, the optimal fermentation temperature was determined to be 37 °C, with a compound ratio of 4:4:3.

3.2.1. Results of Texture Measurement

The quality and stability of yogurt are closely related to its texture. Using Group D-1 and Group E-1 as controls, the texture of the mixed yogurt (B-1) was measured, including its hardness, springiness, cohesiveness, chewiness, and resilience.

Hardness reflects the curd characteristics of yogurt, with higher hardness indicating greater morphological stability [35]. Significant differences in hardness were observed among the different yogurt groups (p < 0.05) (

Table 6. Yogurt texture measurement results.

Sample	Hardness (g)	Springiness	Cohesiveness	Chewiness	Resilience
B-1 (1)	830.78 ± 29.48 a(2,3)	0.98 ±0.00 b	0.98 ± 0.00 a	844.43 ± 23.62 a	0.94 ± 0.02 ab
D-1	246.77 ± 17.04 c	1.17 ± 0.06 a	0.95 ± 0.00 b	266.02 ± 30.93 b	0.93 ± 0.01 b
E-1	725.45 ± 44.73 b	1.04 ± 0.04 b	0.98 ± 0.00 a	813.49 ± 21.43 a	0.96 ± 0.00 a

⁽¹⁾ As presented in Table 4. ⁽²⁾ The results present the mean ± standard deviation (n = 3). ⁽³⁾ Lowercase letters (a, b, c, etc.) are used to indicate significant differences, with different letters within the same column representing statistically significant differences (p < 0.05), while the same letters denote no significant difference.

). Group B-1 exhibited the highest hardness, while Group D-1 had the lowest. In terms of springiness, the differences among the groups were not significant, with Group D-1 showing the highest elasticity, followed by Group E-1. To some extent, cohesiveness can reflect the texture of yogurt; higher cohesiveness indicates a more uniform and delicate texture [36]. Groups B-1 and E-1 demonstrated higher cohesiveness, chewiness, and resilience compared with Group D-1, with Group B-1 achieving the highest value in chewiness. Therefore, it can be inferred that the addition of protease-producing Lactiplantibacillus plantarum NH-24 increases the hardness, cohesiveness, and chewiness of yogurt, potentially improving its texture and enhancing its chewiness.

3.2.2. Results of Volatile Flavor Compounds Measurement

Flavor compounds are a critical indicator for assessing dairy products, as their diversity can impart unique aromas and tastes. The volatile flavor compounds in the mixed yogurt were analyzed using GC-IMS technology.

A total of 55 signal peaks were detected across the three groups of mixed yogurt. Qualitative analysis of their volatile components identified 14 ester compounds, 12 ketone compounds, 9 alcohol compounds, 6 aldehyde compounds, 2 acid compounds, and 12 other compounds (

Table 7. Qualitative results of volatile compounds.

Number	Compound Name	RI	Rt (s)	Dt (a.u.)	Odor Characteristics.
Ester compounds
1	Butyl 2-methylbutanoate	1051.6	416.307	1.37854	Fruity, herbal aroma
2	Ethyl formate	823.7	243.2	1.06871	Fragrant aroma
3	Hexyl formate	1366.1	946.007	1.33041	Fruity fragrance
4	Acetic acid ethyl ester(M) (1)	898.9	286.722	1.09947	Fresh and fruity aroma
5	Acetic acid ethyl ester(D) (2)	900	287.44	1.33458	Fresh and fruity aroma
6	Ethyl propanoate	943	315.818	1.14377	Sweet fruit flavor, rum
7	Diethyl malonate	1087.3	461.948	1.24791	Sweet fruit flavor
8	Isobutyl butyrate	929.3	306.497	1.8129	Sweet fruit flavor
9	3-Methylbutyl butanoate	1290.4	800.38	1.40099	Fruity flavor
10	Butyl pentanoate	1296.2	810.315	1.40414	Tropical fruit aroma
11	Methyl 2-nonynoate	1299.8	816.938	1.97685	Floral fragrance
12	Butyl 3-methylbutanoate	1078.2	449.796	1.89749	Apple flavor, sweet pineapple flavor
13	2-Methylpropyl 2-methylpropanoate	1092.2	468.545	1.80236	Tropical fruit flavor
14	Propyl thioacetate	1194.3	656.808	1.554	Garlic flavor
Ketone compounds
15	2-Butanone	915.7	297.498	1.24088	Minty flavor
16	2-Pentanone	999.8	358.205	1.37036	Sweet fruit aroma, wine aroma
17	2-Hexanone	1098.2	476.878	1.18988	Fruity flavor
18	2-Heptanone(M)	1193.8	656.176	1.2617	Coconut, herbal aroma
19	2-Heptanone(D)	1192.8	654.836	1.63081	Coconut, herbal aroma
20	2-Butanone, 3-hydroxy(M)	1299.5	816.275	1.06336	Buttery flavor
21	2-Butanone, 3-hydroxy(D)	1294.7	807.665	1.32841	Buttery flavor
22	4-Methyl-2-pentanone(M)	1004.3	362.848	1.17824	Pleasant scent
23	4-Methyl-2-pentanone(D)	1025.9	386.432	1.478	Pleasant scent
24	2-Propanone	843.3	253.834	1.11587	Oily, diffusive aroma
25	Cyclopentanone	774.8	218.461	1.10457	Minty flavor
26	1 -Hydroxy-2-propanone	1294.1	806.672	1.2369	Caramel aroma
Alcohol compounds
27	1-Propanol	1053.1	418.174	1.1113	Fruity aroma, wine aroma
28	1- Butanol(M)	1158.2	583.47	1.38377	Wine scent
29	1- Butanol(D)	1158.9	584.869	1.17909	Wine scent
30	1-Propanol, 2-methyl(M)	1107.6	492.18	1.16998	Nutty flavor, camphor aroma
31	1-Propanol, 2-methyl(D)	1107.6	492.18	1.36729	Nutty flavor, camphor aroma
32	1-Butanol, 3-methyl	1193.6	655.943	1.49732	Whiskey flavor
33	2- Butanol	1037.3	399.404	1.14923	Wine fragrance
34	Tetrahydrolinalool	1099.1	478.371	1.2761	Fruity aroma
35	Cis-2-pentenol	1344.4	901.718	1.44383	Fruity aroma
Aldehyde compounds
36	Trans,trans-2,4-hexadienal	1363.2	939.88	1.11111	Sweet taste, floral aroma
37	3-Methyl-2-butenal	1212.6	682.09	1.09159	Sweet fruit aroma
38	Propanal	825	243.896	1.14493	Nutty flavor
39	1-Hexanal(M)	1053.2	418.272	1.25698	Grassy scent, apple aroma
40	1-Hexanal(D)	1051.2	415.913	1.55536	Grassy scent, apple aroma
41	Trans-2-Heptenal	928	305.654	1.66798	Grassy flavor
Acid compound
42	Acetic acid(M)	1493.2	1253.461	1.05341	Lactic acid flavor
43	Acetic acid(D)	1492	1250.11	1.16259	Lactic acid flavor
Other compounds
44	Pyridine	1218.6	690.579	1.25047	Pungent odor
45	2-Ethylpyridine	1294.5	807.334	1.09964	Grassy flavor
46	Pyrrolidine	1024.5	384.86	1.28046	Ammonia smell
47	Tetrahydrofuran	899.2	286.96	1.22605	Minty flavor
48	Furan, 2-methyl-3-(methylthio)(M)	1345.7	904.236	1.10467	Minty flavor, spicy taste
49	Furan, 2-methyl-3-(methylthio)(D)	1345.5	903.822	1.15915	Minty flavor, spicy taste
50	Thiazole	1262.2	755.342	1.25584	Nutty flavor
51	Camphene	1051.9	416.7	1.20587	Camphor aroma
52	Trans-β-ocimene	1252.8	740.771	1.20219	Citrus flavor
53	Acrylonitrile	1027	387.612	1.09397	Pungent odor
54	Triethylamine	830.3	246.722	1.46784	Ammonia smell
55	Allyl sulfide	1148.9	565.56	1.11909	Onion flavor, garlic flavor

⁽¹⁾ (M) Monomer. ⁽²⁾ (D) Dimer.

).

Ester compounds are one of the primary contributors to yogurt flavor. They enhance the fresh and fruity aroma of yogurt, primarily formed through the esterification of milk fatty acids with alcohols [37]. During lactic acid bacteria fermentation, the oxidation of unsaturated fatty acids generates numerous ketone compounds, which often possess pleasant odors [38]. Alcohols are produced through chemical reactions such as lactose metabolism, amino acid degradation, and the breakdown of secondary hydroperoxides of fatty acids, imparting fruity and wine-like aromas [29]. The metabolic processes of lactic acid bacteria yield aldehyde compounds, which have low flavor thresholds and contribute to grassy and aromatic notes. Acid compounds help reduce yogurt acidity, inhibit the growth of harmful microorganisms, and are a key source of yogurt’s sour taste. Together, these volatile organic compounds form the distinctive flavor profile of mixed yogurt.

A differential fingerprint map was created to analyze the differences in volatile flavor compounds among the three yogurt groups (Figure 7). On the right side are the sample codes, and the horizontal axis represents the volatile compounds. The color and intensity of the characteristic peaks in the map indicate their relative concentrations. As shown in the figure, region “a” represents the common flavor characteristic peaks shared by all three yogurt groups, with no significant differences in component concentrations. Region “b” also contains common characteristic peaks, including compounds such as hexyl formate and acetic acid (M), but with varying concentrations, where Group E-1 had the highest concentration, followed by Group B-1. Region “c” shows higher concentrations in Groups D-1 and B-1. In region “d”, Groups D-1 and E-1 had lower concentrations, making it primarily a unique flavor characteristic peak region for Group B-1, containing ester compounds such as methyl 2-nonynoate, butyl 3-methylbutanoate, and 2-methylpropyl 2-methylpropanoate, which impart rich fruity notes. Region e includes compounds such as 1-butanol (M), 1-butanol (D), 1-propanol, 2-methyl (M), and 1-propanol, 2-methyl (D), forming the characteristic peak region for Group E-1 and contributing to its wine-like aroma. However, the presence of tetrahydropyrrole and diallyl sulfide also introduced pungent odors. Therefore, it is concluded that Group B-1 exhibits a more balanced odor profile, combining the fresh and fruity notes of Groups D-1 and E-1 while reducing the pungent odors of Group E-1, resulting in higher overall acceptability.

A clustering heatmap depicting the relative content of volatile components in the three yogurt groups is shown in Figure 8. In the heatmap, blue denotes a negative correlation, while red indicates a positive correlation, as illustrated in the color legend on the right. The heatmap provides a clear visualization of the differences in the relative content of various compounds among the three yogurt groups. Based on cluster analysis, the samples can be categorized into two main groups: E-1 forms one distinct cluster, while B-1 and D-1 group together, aligning with the findings from the fingerprint analysis.

3.2.3. Results of the Electronic Nose Analysis

The electronic nose was employed to analyze the three yogurt groups, B-1, D-1, and E-1. Based on the collected data, a radar chart (Figure 9A) and a principal component analysis (PCA) plot (Figure 9B) were constructed.

Among the sensors, LY2/G, LY2/AA, LY2/GH, and LY2/gCTL exhibited negligible responses to the odors of the three yogurt groups, while the remaining 14 sensors demonstrated varying levels of responsiveness (Figure 9A). The highest response value was recorded for P30/2 across all yogurt groups, followed by P30/1 and PA/2, suggesting the possible presence of specific organic compounds in the yogurt. Comparative analysis of the response values revealed that B-1 showed the highest response, with D-1 ranking second, indicating that the blended yogurt had a more pronounced odor intensity compared with the yogurt fermented exclusively with Lactiplantibacillus plantarum NH-24.

In principal component analysis (PCA), the distance between samples serves as a quantitative measure of odor differences [39]. PCA was conducted on the yogurt groups to evaluate their odor characteristics and flavor distinctions. The contribution rates of PC1 and PC2 were 80.7% and 14.8%, respectively, with a cumulative contribution rate of 95.5%, encompassing the majority of the original data and effectively representing the overall sample information (Figure 9B). The close proximity between the D-1 and E-1 groups indicates that they likely share similar flavor profiles, while B-1 is relatively distinct. The observed distances among the three groups suggest variations in the content of flavor compounds.

3.2.4. Results of the Electronic Tongue Analysis

The electronic tongue is capable of assessing the taste profile of yogurt, determining its sensory quality, highlighting variations among different yogurt products, and offering a scientific foundation for quality control throughout the production process [40].

The seven sensors of the electronic tongue were utilized to analyze each yogurt group, revealing distinct response patterns among the three groups (Figure 10A). Group B-1 demonstrated the highest response on the umami and sweetness sensors, while Group E-1 exhibited the strongest response on the sourness sensor. Group D-1 displayed the highest response on the bitterness sensor. Based on a comprehensive analysis, it is likely that Group B-1 yogurt possesses moderate sourness, pronounced sweetness, and mild bitterness, contributing to a more balanced and harmonious flavor profile.

By examining the distribution patterns of each sample group in the figure, the flavor variations in the yogurt can be quantitatively assessed. Distinct differences in taste profiles were observed among Group B-1, Group D-1, and Group E-1, with a notable separation between Group B-1 and Group E-1 (Figure 10B).

3.2.5. Results of Acidity Determination of Yogurt During Storage

During storage, the acidity of yogurt progressively increases as the starter cultures continue to ferment and produce lactic acid. This increase in acidity can impact the yogurt’s flavor, texture, and potentially its consistency and nutritional profile [41]. By conducting acidity measurements during storage and analyzing its variations, the degree of post-acidification and storage stability can be assessed. This information serves as a valuable basis for determining the optimal storage period.

With prolonged storage time, the pH of all yogurt groups decreased, with the most significant decline occurring within the first 0–10 days. Among the groups, D-1 showed the fastest pH reduction rate, followed by B-1, while E-1 exhibited the slowest decline (Figure 11A). Simultaneously, acidity increased over the storage period (Figure 11B). During the 20-day storage period, the acidity of groups B-1, D-1, and E-1 increased by 7.84 °T, 12 °T, and 4.83 °T, respectively. These findings indicate that Lactiplantibacillus plantarum NH-24 may mitigate post-acidification in yogurt, potentially extending its shelf life without adversely affecting its flavor. This highlights the significant potential of Lp. plantarum NH-24 in fermented dairy production.

4. Conclusions

The application of Lactiplantibacillus plantarum NH-24, a protease-producing strain, in yogurt fermentation was evaluated by measuring key parameters such as coagulation time, curd quality, acidity, water-holding capacity, and sensory attributes. The results indicate that the optimal fermentation temperature is 37 °C, with a blending ratio of 4:4:3. Under these conditions, the blended yogurt demonstrated an ideal coagulation time, superior curd quality, moderate acidity, and optimal water-holding capacity. All measured parameters met national standards, and the yogurt received the highest sensory evaluation scores.

Further studies on the fermentation properties of the blended yogurt were carried out. Texture profile analysis demonstrated that incorporating Lp. plantarum NH-24, a protease-producing strain, significantly improved the hardness, cohesiveness, and chewiness of the blended yogurt. These enhancements indicate that the strain has the potential to optimize and refine the texture of yogurt.

Analysis of volatile flavor compounds identified a total of 13 esters, 9 ketones, 7 alcohols, 5 aldehydes, 1 acid, and 11 other compounds across the three yogurt groups. The findings suggest that Group B-1 displayed a more balanced aroma profile, integrating the fresh and fruity notes characteristic of Group D-1 and Group E-1, while mitigating the pungent odor associated with Group E-1.

Analysis of the electronic nose results demonstrated that the blended yogurt containing Lp. plantarum NH-24, a protease-producing strain, displayed a stronger aroma intensity. Electronic tongue analysis further revealed that the blended yogurt exhibited the highest responses on the umami and sweetness sensors, along with moderate acidity and the lowest bitterness. These results suggest that the incorporation of Lp. plantarum NH-24 has the potential to improve the flavor characteristics of the yogurt.

Analysis of the acidity measurement results during storage indicated that incorporating Lp. plantarum NH-24, a protease-producing strain, significantly slowed the rate of acidity increase in yogurt. This demonstrates that the strain has the potential to alleviate post-acidification during storage, thereby enhancing the stability and quality of yogurt over time.

In conclusion, incorporating Lp. plantarum NH-24, a protease-producing strain, into yogurt fermentation holds promise for improving flavor characteristics and extending shelf life, offering a scientific foundation and practical reference for its application in yogurt production. However, further in-depth studies are required to address the preparation of this strain as a starter culture, including optimizing harvest timing, centrifugation conditions, and inoculation levels, which are essential for scaling up to industrial production.