Fengxun Lng, Jinzhu Wen, Zhen Wu,*, Dodong Pn,b,*, Linjun Wng

a Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, School of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315211, China

b School of Food Science & Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210097, China

c Qingyunshan Pharmaceutical, Guangdong, Guangzhou 512000, China

Keywords:

Lactobacillus plantarum

Flavor profiles

Volatile organic compounds

Sensory profiles

Fatty acid

A B S T R A C T

Lactic acid bacteria (LAB) fermentation is the simplest and safest way of food preservation, and the use of probiotics in yoghurt could provide dairy products with unique flavors, textures and health benefits. In this study, Lactobacillus bulgaricus, Streptococcus thermophilus, L. reuteri DSMZ 8533 and the potential probiotic strain L. plantarum A3 were used for the milk fermentation. Results found the texture properties such as hardness, consistency, and viscosity of the yoghurt were enhanced in the mixed culture condition.Furthermore, components like amino acid (leucine), vanilla (vanillin), C18:3n6 (unsaturated fatty acids) were also accumulated in L. plantarum A3 fermented yoghurt, which leads to the significant sensory pro filing difference compared with the former plain yoghurt. All these results proved L. plantarum A3 is a potential probiotic strain which could enhance the sensory and nutrition pro filing of the fermented milk. Future work still needs to be done on the synergistic interaction between the traditional strains and the probiotics during the fermentation process.

1. Introduction

Fermented food has been used in human diet for thousands of years and the use of microbial starters could provide the products with unique flavors and textures properties. In recent decades, the associated health benefits and functions of active free amino acid,peptides and vitamins produced during the fermentation process are attracting attention worldwide [1,2]. Lactic acid bacteria (LAB)fermentation is also the simplest and safest way of food preservation,since the lactic acid produced by LAB can extend the shelf life of food and provide beneficial effects to humans by regulating the microecological balance of the gastrointestinal tract (GIT) [3,4]. Some fermented milk products with functional milk casein hydrolysate have already been used in sports beverage, which can promote cardiovascular improvement after the proper physical exercise [5].

Besides dairy products, most health promoted probiotics belong to lactic acid-producedLactobacilliandBifidobacteriastrains, which exist in more than 90% of the food products like yoghurt, fermented pickles or other fermented foods [6,7]. As an important member of LAB,L. plantarumis usually found in plant-based fermented food and the potential probiotic characters have also gained more interest in different products [8,9]. With the increasing commercial need of the probiotic bacteria in fermented dairy industry, a wide variety of probiotic LAB strains are also available and co-fermented in the dairy products. Furthermore, the success of new functional foods depends not only on the enhanced nutritional value, such as the vitamins and active peptides, but also on the ideal sensory qualities which meet the needs of consumers [10]. Though the bacterial co-culture fermentation model could produce some special volatile flavor compounds during the fermentation process, the quality of the fermented milk and the degrees of kinetic acidification parameters were affected by the addition of prebiotics and probiotics in the co-culture fermentation system [11]. Some studies also found that in the processing of Cheddar cheese co-cultured with probiotics, there were significant differences in protein hydrolysis, resulting in higher concentrations of free amino acids (FAAs) and acetic acid in organic acids in probiotic cheeses [12].

Different combinations of LAB starters provide the fermented dairy products with potential nutrition and health benefits. However,either beneficial or undesirable changes may also be generated in the bacterial flora during the fermentation process. The aim of this work was to analyze the interactions among LAB starters with the GIT tolerance strain ofL. plantarumA3, and the texture and flavor characteristics of the yoghurts fermented withL. plantarumA3 were also investigated for the development of probiotic dairy products.

2. Materials and methods

2.1 Strains and culture conditions

The 16s rRNA method was used to identify theLactobacillusstrains isolated from traditional Sichuan pickles [13], stored in-80 °C refrigerator, and further activated and cultured in 37 °C MRS broth.Streptococcus thermophilusABT-T (ATCC 43077),L. bulgaricusKLDS1.0207 (CICC No.20247) andL. reuteriDSMZ 8533 were purchased from China General Microbial Culture Collection Center (Beijing, China). Raw milk with the fat content of 3.2% was purchased from Ningbo Dairy Group Co.,Ltd., (Ningbo, China).

2.2 Growth characteristics of L. plantarum A3 in mixedculture condition

Exploring the effects ofL. plantarumA3 under mixed-culture condition,L. plantarumA3,L. bulgaricusandS. thermophiluswere cultured in 37 °C MRS medium under the same conditions with different inoculation ratios. The growth of the strain under the mixed strain culture conditions was measured at 600 nm every 2 h, and the pH value of the acid production was measured with a digital pH meter within 24 h culture time.

2.3 Probiotic analysis of LAB strains

Probiotic analysis was performed onL. bulgaricus,S. thermophilus,L. plantarumA3 andL. reuteri. The self-aggregating ability of lactic acid bacteria was slightly changed according to the method of Del Re et al. [14]. Mucin adhesion test mainly refers to the method of Izquierdo et al. [15]with some modifications. Tolerance analysis of bacteria in stimulated gastrointestinal fluid was also carried out according to our previous study [16]. During all the analysis, the OD values of the single and co-culture strains were 1.0 at 600 nm.

2.4 Fermentation characteristics of the yoghurt

The OD value of the strain was adjusted to 1 ±0.02 before the fermentation process. During the milk fermentation, fresh pasteurized milk was inoculated with 2% (V/V) OD-adjusted bacterial solution at 37 °C, and the ratiosof the starters (L. bulgaricus:S. thermophilus:L.plantarumA3) were 1:1:0, 1:1:1, 1:1:2, 1:1:3, the viability count of different starters was all 108CFU/mL. Fermented at 37 °C for 5 h,then ripening at 4 °C for 24 h before further analysis. The firmness,consistency, cohesiveness and index of viscosity of the yoghurt were determined by using a texture analyzer (TA, UK), and the cylindrical probe procedures are as follows (40 mm cylinder probe): 70 mm height, 65 mm drop, 3 mm/s descent speed, 5 g contact force; return distance 70 mm, return speed 10 mm/s. Meanwhile, the measuring cup and cylinder probe should be cleaned after each test.

Titratable acidity of the yoghurt was determined with the base titration method. After sample homogenization, two drops of phenolphthalein were added to 1 g sample (diluted to 10 mL with ddH2O) and then titrated with 0.1 mol/L NaOH (V0.1), and the result was expressed as Thorner degrees (°T), calculated as 10 FV0.1·°T, F is the correction factor.

2.5 β-Galactosidase and lactate dehydrogenase analysis

Cells in the co-cultured conditions were suspended in PBS buffer and hydrolyzed by lysozyme at 37 °C for 1 h [17]. After that, the crude enzyme solution was dissolved in 0.4 mol/L NaCl solution before the enzyme activity analysis. The activity ofβ-galactosidase and lactate dehydrogenase under mixed-culture condition were assayed according to the assay kit and the method of Vasiljevic et al. [18].

2.6 GC-MS analysis of volatile compounds

The fermented milk (5 mL) was sealed in a 20 mL headspace vial (CNW Technologies, Germany) with a Teflon/silicone septum in an aluminum cap, and 10 μL 2-methyl-3-heptanone (10 ng/μL)was added as an internal standard. Extraction of volatile compounds by Solid Phase Micro extraction (SPME) with 75 μm Carboxy/Polydimethylsiloxane (CAR/PDMS) fibers from Supelco Inc(Bellefonte, PA, USA). The constant flow rate of the carrier gas(helium) was 3 mL/min, with an inlet temperature of 210 °C and split less injection mode. The initial temperature of the column oven was 40 °C, which was maintained for 3 min, increased to 140 °C at 4 °C/min, held for 1 min, and then increased to 200 °C at 10 °C/min for 20 min. The signal acquisition of the mass spectra was in full scan mode, ionization method EI, electron bombardment energy of 70 eV,interface temperature of 220 °C, ion source temperature of 230 °C,quadrupole rod temperature of 150 °C, scan mass range of 40-600m/z,and scan frequency of 3.6 scans/s.

2.7 Fatty acid composition analysis

The fermented milk sample was mixed with CH3OH/CH2Cl2(1:3)for 10 min at room temperature and oil components were ultrasonic extracted after centrifugation procedure (1 800 r/min, 10 min). The supernatant was blow-dried with nitrogen and dissolved in methanolic KOH (6% ) followed by 4 mol/L HCl. After BF3-MeOH treatment,fatty acid compositions were extracted withn-hexane for 3 times,and the extraction solution was transferred to 2 mL sample bottle and blow-dried with nitrogen for further analysis.

The fatty acid composition was analyzed using a TG-5MS 30 m × 0.25 mm × 0.25 μm column, the heating procedure as follows:starting temperature at 80 °C, maintaining for 1 min, then raising the temperature to 200 °C at 10 °C/min, after that, continue raising the temperature to 250 °C at a rate of 5 °C/min, and finally keeping the temperature at 270 °C. During the sample loading procedure, the sample extract injection volume was 1 μL, and the flow rate of carrier gas helium was 1.2 mL/min. The Scanning range of the GC-MS was 30-400m/z. The content was calculated by external standard method and internal standard method, and all the data were collected from 3 replicates samples.

2.8 Amino acid analysis

The homogenized fermented milk samples were vapor phase hydrolyzed with 6 mol/L HCl solution in a drying oven at 110 °C for 24 h. At the end of the hydrolysis, samples were kept in the room temperature and neutralized with 4 mol/L NaOH, finally dried in a speed vacuum centrifuge. Before the amino acid analysis, citric acid buffer solution was used to solubilize amino acids prior to filtering.The amino acid profiles of the different samples were determined by L-8900 amino acid automatic analyzer (Physics and Chemistry Test Center, Jiangsu Province, China). The amino acid solution was loaded on the ion exchange column (4.6 mm × 60 nm, 3 μm) with a flow rate of 0.45 mL/min at 57 °C, followed by post-column derivatization(135 °C) with ninhydrin. The concentration of the amino acid was calculated according to the colorimetric detection at 420 nm.

2.9 The E-tongue analysis

The Alpha Astree II potentiometric electronic tongue system(Isenso, New York, NY) contains a sensor array of 6 working electrodes, with a platinum column electrode as an auxiliary electrode and Ag/AgCl as a reference electrode. The working electrode was subjected to multi-frequency large-amplitude pulse voltammetry scanning of the solution one by one. Before data collection, the detection head was cleaned for 2-3 min with distilled water. The prepared fermented milk samples were homogenized, poured into an electronic tongue special cup and data were collected at room temperature. For all the samples, 5 replicates were carried out and 3 stable data were collected for principal components analysis(PCA) and orthogonal partial least squares discrimination analysis(OPLS-DA). Standard solutions of 0.01 mol/L hydrochloric acid were used to prepare the system according to the manufacturer’s instructions.

2.10 Data analysis

All statistical analysis was performed using SPSS (SPSS Inc./IBM Corp., Chicago, IL) and the plots were drawn with Origin 8.5 (Origin Lab, Northampton, MA) software. For visualizing the concentration distribution of the yoghurt component with microarray heatmap,data were analyzed with Multi-Experiment Viewer (MeV) software.Furthermore, SIMCA-P software was also adopted for the OPLSDA model establishment with variance testing of cross-validated predictive residuals (CV-ANOVA,P＜ 0.05) and permutation testing.PCA and variable importance for the projection (VIP) predictive and orthogonal analysis were used in SIMCA-P software.

3. Results

3.1 The probiotic properties and growth characteristics of L. plantarum A3

L. plantarumA3 has higher auto-aggregation and adhesion activity, compared with other fermented strains, such asL. reuteri,L. bulgaricusandS. thermophilus(Figs. 1A, 1B). Meanwhile, the viability ofL. plantarumA3 after gastrointestinal fluid treatment was also higher than other single strains in this study (Fig. 1C). WhenL. plantarumA3 added in the mixed culture condition, the viability of the mixed strains was higher than theL. plantarumA3 free groups(Fig. 1D). Furthermore, it demonstrates thatL. plantarumA3 can promote the increase of cell density and the medium pH also found sharp decrease under mixed-culture condition (Figs. 1E, 1F).

Fig. 1 Probiotic properties of L. plantarum A3 in the co-cultured condition and the fermentation growth kinetics of mixed-culture strains. Auto-aggregation (A)and adhesion (B) rate of different strains; number of the live bacteria in the single (C) and co-cultured condition (D); OD values (E) and pH changes (F) of the strains in different inoculation ratios during the 24-h culture time.

3.2 Texture changes of the yoghurt with different starters

As shown in Fig. 2,L. plantarumA3 has a good performance on the physical quality of fermented milk. Along with the increased proportion ofL. plantarumA3, the hardness, consistency, viscosity,and viscosity coefficient were also changed with the ratio ofL. plantarumA3. In terms of acidity and water holding capacity on the texture formation of yogurt, no significant effects were found between the different groups.

Fig. 2 Texture, water holding capacity and acidity evaluation of the yoghurt fermented with different strains. The ratio of L. bulgaricus:S. thermophilus:L. plantarum A3 were 1:1:0, 1:1:1, 1:1:2, 1:1:3, respectively.

3.3 β-Galactosidase and lactate dehydrogenase analysis

The activity ofβ-galactosidase and lactate dehydrogenase in culture media supernatant with different ratio ofL. plantarumA3,L. bulgaricusandS. thermophiluswere detected in the stationary phage (18 h). The activity ofβ-galactosidase decreased gradually when the proportion ofL. plantarumA3 increased. Before 1:1:2, the activity of lactate dehydrogenase increased with the increase of the ratio ofL. plantarumA3. When the ratio was 1:1:3, the activity of lactate dehydrogenase decreased compared with 1:1:2 (Fig. 3).

3.4 GC-MS analysis

Fermented milk contains a complex mixture of volatile organic compounds (VOCs), however, only small parts of the VOCs have the essential effects on the flavor of the yoghurt. In this study, we used SPME-GC-MS to identify the VOCs in fermented milk with different starters. It showed that milk fermented withL. plantarumA3 was significant different fromL. plantarumA3 free groups, and the violatile flavor components in theL. plantarumA3 group were relatively close to each other (Fig. 4A). As shown in Fig. 4B, the concentration of styrene, acetic acid, benzene and 2-heptanone are higher in theL. plantarumA3 fermented group, and part of the high concentration VOCs, such as 2-heptanol and acetic acid (acidic flavor)also showed the positive effect on the main components of the VOCs.Some components, like butanoic acid (aromatic flavor), though has a relative lower concentration (VIP ＞ 1), still has the positive effect on the VOCs (Fig. 4C).

Fig. 4 Volatile flavor profiles of the fermented milk with different proportion of L. plantarum A3. Wherein A1, A2 and A3 belong to the 1:1:0 group; B1-1, B1-2 and B1-3 belong to the 1:1:1 group; B2-1, B2-2, and B2-3 belong to the 1:1:2 group; B3-1, B3-2, and B3-3 belong to the 1:1:3 group. (A) PCA analysis of volatile flavors between different groups; (B) two-way hierarchical cluster dendrogram and heatmap visualization of the volatile flavors; (C) VIP plot of OPLS-DA model in violate flavor components (VIP (2+5+0) means predictive analysis, both the PC1 and PC2 were analysis in all the 5 groups).

3.5 Amino acid and fatty acid changes

The amino acid content of the fermented milk in different groups was analyzed with the PCA method. As shown in Fig. 5A,L. plantarumA3 fermented yoghurt groups are relatively closer and the concentration of related amino acids (histidine,methionine, tyrosine, glycine) were also increased during the milk fermentation processing compared with control group(Fig. 5B). Meanwhile, with the VIP analysis, amino acids such as histidine, isoleucine, leucine, valine, methionine, tyrosine,glycine are the main positive amino acids inL. plantarumA3 fermented group (Fig. 5C).

Fig. 5 The changes of free amino acids in different fermented milk groups. (A) PCA analysis of the amino acids in the different fermented milk group.(B) Heatmap visualization of the amino acid components; (C) VIP plot in 1:1:1 group (VIP (1+3+1) means VIP orthogonal analysis, only the PC1 was analysis in the three 1:1:1 group sample).

The changes of fatty acid composition in yoghurt fermented by different proportions ofL. plantarumA3 were analyzed by GC-MS.It can be seen from the PCA analysis in Fig. 6A that all the groups have the similar principal components, indicating that the addition ofL. plantarumA3 has little effect on the composition changes of the fermented milk fatty acid. The groups withL. plantarumA3 was relatively closer compared with the control group (Fig. 6B),and C20:1, C18:3n6, C15:0, C18:3n3, C17:0, C22:2, and C13:0have relatively large contributions to the fatty acid profiles in theL. plantarumA3 fermentation groups (Fig. 6C). Meanwhile,L. plantarumA3 can promote the increase of then-3 fatty acid during the yoghurt formation, and the concentrations of these kinds of fatty acid are significantly different among theL. plantarumA3 fermented groups(Table 1).

Fig. 6 PCA analysis of the changes of the fatty acids in different samples. (A) PCA analysis of fatty acid in different groups, where control is unfermented milk,A is 1:1:0 group, B1 is 1:1:1 group, B2 is 1:1:2 group, B3 is 1:1:3 group. (B) Heatmap visualization of the fatty acids in different samples; (C) VIP plot of all the fatty acids groups (VIP (2+5+0) means predictive analysis, both the PC1 and PC2 were analysis in all the 5 groups).

Table 1Differences of the unsaturated n-3 or n-6 fatty acids in different fermented milk samples (mg/kg).

3.6 Electronic tongue analysis

The results of the electronic tongue analysis (Fig. 7) are similar with the data from volatile flavor and amino acid analysis,and samples with different strains had significant difference which can be easily identified. According to the results, PC1 contributed 47.11% of the total variance and PC2 contributed 18.09% , for a cumulative contribution of 65.2% , which represent most of the information in the whole sample. Samples with linear discriminant analysis (LDA) showed the similar trends with the PCA analysis.The discriminant index for the PCA and LDA are 98.47% and 99.96% , respectively.

Fig. 7 Electronic tongue analysis of the fermented milk with and without L. plantarum A3 strain. PCA (A) and LDA (B) of the main taste compounds in the control (unfermented) and fermented milk groups(1:1:0, 1:1:1, 1:1:2, 1:1:3).

4. Discussion

Probiotic yogurt has many health benefits, as it contains a reasonable number of probiotics which can help in maintain microbial flora balance in human GIT.L. plantarumA3 isolated from Sichuan pickle product, reveals great potential in probiotic yogurt in terms of bacterial population, adhesion, auto-aggregation properties in both previous [13]and this study. Meanwhile, the texture and flavor characteristics, including consistency and VOCs, were enhanced when combined with the traditionalL. bulgaricusandS. thermophilusstrains.

In this research,L. plantarumA3 co-cultured with traditional fermentation bacteria has a good lactic acid producing ability,which could short the curding time, improve the sensory profiling of the yoghurt. Similar study also found that during the mixed culture condition,L. bulgaricuscan promote the growth and acid reproduction ofS. thermophilusduring milk fermentation process [19].Lactose in milk can be converted into lactic acid during fermentation,andβ-galactosidase and downstream lactate dehydrogenase are responsible for this conversion [20,21]. Though activity ofβ-galactosidase in the 1:1:1 group was similar with the control group,the activity of lactate dehydrogenase was much higher than the control group, which enhanced the lactic acid production from the transformation of pyruvate. This is also the reason why the titratable acidity and pH value of theL. plantarumA3 ratio of 1:1:1 group is much higher than other groups, and the strains ratio of 1:1:1 is the best for the mixed-strain starter in milk fermentation.

Yoghurt fermentation with probiotics indeed has a good quality and nutrition properties compared with the traditional strains based on their safety, nutritional value and health-promoting properties.Some studies have shown that the addition ofLactobacillusandBifidobacteriacan increase the concentrations of FAAs in all probiotic cheeses [22]. In general, the VOCs compounds presenting in the fermented yoghurt products are from the degradation of casein and side chain modifications, including keto acids, ammonia, amines,aldehydes, acids and alcohols, which are the important contributors to the flavor profile of yoghurt [23]. The difference between theL. plantarumA3 and theL. plantarumA3-free yoghurt on the content of free amino acid components and the main flavors based on the PCA analysis indicated that the addition ofL. plantarumA3 indeed can enhanced the sensory pro filing of the yoghurt, especially components like vanilla (vanillin), leucine, C18:3n6.

According to the recent study, several non-nutritional components such as sphingolipids in yoghurt, conjugated linoleic acid (CLA)and butyric acid can act as anticancer agents [24]. Some studies have shown that fermented dairy products contain higher levels of CLA than non-fermented milk [25]. In this study,L. plantarumA3 can enhance the level of omega-3 fatty acids profile in the fermented milk,unsaturated fatty acids (C18:3n6, C18:3n3) account for a large proportion of yoghurt, wherein the first main component and the second main component mainly contain unsaturated fatty acids. It also revealed that the fatty acid composition involved in the inflammatory response of the cell andn-3 PUFAs have the potential anti-inflammatory effects which may be useful as therapeutic agents in the treatment of GIT disorders [26]. This may be the reason why yoghurt consumption can modulate the immune system and prevent the inflammatory process in a mouse model [27].

5. Conclusions

L. plantarumA3, as the plant-origin lactic acid bacteria, has potential probiotic characteristics in terms of bacterial population,adhesion, auto-aggregation properties in this study. Meanwhile,the sensory and nutrition profiling of the fermented milk withL. plantarumA3 were also enhanced by increasing the contents of amino acids and volatile compounds and unsaturated fatty acids.However, future work will need to focus on the interaction between the traditional fermentation strains and probiotics in the mixed probiotic yoghurt products, in order to meet the quality and nutrition requirements of consumer.

Ethics statements

Our research did not include any human subjects and animal experiments.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [32072192, 31901668, 31671869], Key Research and Development Project of Zhejiang Province [2020C02042], the Natural Science Foundation of Zhejiang Province [LY19C200005],the Natural Science Foundation of Ningbo (202003N4129), the Open Project Program of the First-Class Bioengineering Disciplines in Zhejiang Province [KF2020007], the Graduate General Program of the Education Department in Zhejiang Province [Y202045625]and the K. C. Wong Magna Fund in Ningbo University.

Conflict of interest

The authors declare no conflict of interests.