Characterization of the Lactobacillus plantarum A3 with novel fermentation properties

The use of probiotic starters in the milk fermentation could provide the dairy products with unique flavors, textures and some health benefits. In this research, L. plantarum A3 was identified as a probiotic with good fermentation characters during the texture and flavor formation of yoghurt. The hardness, consistency, viscosity, and viscosity coefficient of the yoghurt were enhanced when fermented with L. plantarum as the higher activity of β-galactosidase and lactate dehydrogenase in the mixed culture condition. The contents of amino acids and volatile organic compounds (VOCs) were found accumulated in L. plantarum fermented yoghurt besides the butanoic acid, acetone. Sensory profiling evaluation with electronic tongue system proved the novel taste of L. plantarum fermented yoghurt were significant different with the former plain yoghurt. Indeed, yoghurt fermentation with L. plantarum A3 indeed has a good quality and nutrition properties compared with the traditional strains and the relationship between the traditional fermentation strains and probiotics still need further investigation to meet the nutrition requirements of varies consumers.


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, textures and some health benefits, including lowering the risk of high blood pressure, diabetes, obesity, high cholesterol, diarrhea, thrombosis [1]. Meanwhile, fermented products can also provide consumers with good source of free amino acids and vitamins which is amplified in the fermentation process [2,3]. It has recently been shown that Lactobacillus helveticus fermented milk can be used in sports beverage, which could promote cardiovascular improvement during the proper exercise process [4].
Lactic acid bacteria (LAB) fermentation is the simplest and safest way of food preservation and the lactic acid produced by LAB can extend the shelf life of food and provide beneficial effects to humans by regulating the micro-ecological balance of the gastrointestinal tract [5]. Fermented milk products have been consumed in world for perhaps 4000 years and many of the products are derived from the spontaneous LAB fermentation of milk that during the milk preservation process in the local traditional methods [6].
Besides yoghurt products, fermented vegetable products, like pickles and sauerkraut are also popular in the world. As the important member of LAB, L.
plantarum is always found in fermented plant-based foods. As a very flexible and versatile microorganism, the interest towards L. plantarum has increased in recent years, especially in relation to its probiotic potential [7] and its possible application in different fermented foods and beverages [8].
As an increasing commercial interest in the addition of probiotic bacteria to fermented dairy products, a wide variety of probiotic LAB strains are commercially available and co-fermented in the dairy products. The fact is that bacterial coculture does affect the production of characteristic volatile flavor compounds during the fermentation process, and the success of new functional foods depends not only on the enhanced nutritional value, but also the ideal sensory quality attributes that meet consumer needs [9]. Furthermore, the quality of the fermented milk is subject to co-culture ingredients and selection of probiotics, the degrees of kinetic acidification parameters were affected by the addition of prebiotics and probiotics in the yoghurt co-culture system [10]. Some studies also found the concentration of acetic acid and free amino acids in organic acids in probiotic cheese was higher, and there was a significant difference in proteolysis of three batches of cheddar cheese co-cultured with different probiotics [11].
Different combinations of LAB starters provide the fermented dairy products with potential nutrition and health benefits. However, either beneficial or undesirable changes may also generate in the composition of the bacterial flora during the fermentation process of dairy products. The aim of this work was to analyze the interactions among LAB starters with the probiotic L. plantarum A3, the texture and nutrition characteristics of the yoghurts fermented with this novel strain were also investigated in order to get the adequate combinations of strains for the development of probiotic dairy products.

Strains and culture conditions
Lactobacillus strains isolated from traditional Sichuan pickle were identified by the 16s rRNA methods and further culture in MRS broth. The other two traditional strains S. thermophilus and L. bulgaricus were also stored and cultured in our laboratory.

Symbiotic growth characteristics of L. plantarum
Exploring the effects of L. plantarum under symbiotic culture, L. plantarum and L. bulgaricus and S. thermophilus were cultured in 37°C MRS medium under the same conditions with different strain inoculation ratio. Growth and acid production of the strains in the symbiotic growth media were measured at 600 nm every 2 hours and the curves were plotted in 24 hours' culture time.
Fermented milk preparation

Texture, acidity, water holding capacity analysis
The firmness, consistency, cohesiveness and index of viscosity of the yoghurt were determined by using a texture analyzer (TA, UK), and set the cylindrical probe: 70mm height, 65mm drop, 3mm/s descent speed, 500 PPS speed, 5g contact force; return distance 70mm, return speed 10mm/s. The measuring cup and probe should be cleaned after each test. Acidity of the yoghurt was performed with the acid-base titration method: Two drops of phenolphthalein were added to a 10 g sample and then titrated with 0.1 mol/L NaOH. The data were recorded and the acidity was calculated according to the following formula.

GC-MS analysis of volatile compounds
The fermented milk was sealed in a 5ml to 20mL headspace vial (CNW Technologies, Germany) with a Teflon/silicone septum in an aluminum cap. 10 μL of 10 ng/μL 2methyl-3-heptanone was added as an internal standard. Extraction of Volatile

Fatty acid composition analysis
The fermented milk sample were mixed with methanol/CH 2 Cl 2 (1:3) for 10 min at room temperature and oil components were ultrasonic extracted after centrifugation procedure (1800 rpm, 10 min). The supernatant was blow-dried with nitrogen and dissolved in methanolic KOH (6%) followed by 4N HCl. After BF 3 -MeOH treatment, fatty acid compositions were extracted with n-hexane for 3 times and the extraction solution was transferred to 2ml sample bottle, and blow-dried with nitrogen for further analysis.
The fatty acid composition was analyzed using a TG-5MS 30m×0.25mm×0.25μm column, the heating procedure as follows: starting temperature at 80℃, maintained for one minute, then the temperature rises to 200℃ at 10℃/min, 225℃ at 5℃/min, and 250℃ at 2℃/min for 5min. Flow rate is 1.2 mL/min, with helium as the carrier gas. Scanning range 30-400, injection volume 1 μL. The content was calculated by external standard method and internal standard method, and all the samples were done in triplicate. 8

Amino acid analysis
The homogenized fermented milk samples were hydrolyzed with 6 H HCl solution in a drying oven at 110 ° C for 22 hours. The next day, the hydrolyzed sample was filtered, dried and dissolved with 1 ml citric acid buffer solution (pH 2.2). Finally, 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).

Sensory profiling evaluation
The Alpha Astree II potentiometric electronic tongue system (Isenso, New York, NY) with an Ag/AgCl electrode was adopted for the sensory profiling analysis. The prepared fermented milk samples were homogenized and poured into an electronic tongue special cup and data were collected at room temperature. For all the samples, five replicates were carried out and three stable data were collected for principal components analysis (PCA) and discriminant factor analysis.

β-galactosidase and lactate dehydrogenase analysis
Cells were suspended in PBS buffer and lysozyme was added and bathed at 37 ° C for 1 h. After that, the crude enzyme solution was dissolved in 0.4 M NaCl solution before the enzyme activity analysis. The activity of β-galactosidase and lactate dehydrogenase under symbiotic culture conditions were assayed according to the assay kit and the method of Vasiljevic [21].

Data Analysis
All statistical analyses were performed using SPSS (SPSS Inc./IBM Corp., Chicago, IL) and the plots were drawn with Origin 8.5 (Origin Lab, Northampton, MA) software.
The differences between the mean values of the groups were analyzed using a oneway ANOVA with Duncan's multiple range test and P-values < 0.05 were considered 9 statistically significant.

Symbiotic growth of L. plantarum
The growth characteristics of the complex strains (L. plantarum, L. bulgaricus and S. thermophilus) in the 24 hours culture time were obtained, which is shown in Figure   1. When the proportion of L. plantarum increasing in the culture media, the cell density increases significantly with the culture time, and the pH also found sharp decrease with the time. It demonstrates that L. plantarum can promote the increase of cell density and the acid production capacity of the culture strains under symbiotic condition.

Texture changes of the yoghurt with different starters
As shown in Figure 2, L. plantarum A3 has a good performance on the physical quality of fermented milk. Along with the increased proportion of L. plantarum A3, the hardness, consistency, viscosity, and viscosity coefficient were great changed with the ratio of L. plantarum A3. In terms of acidity and water holding capacity, L.
plantarum A3 also showed a relatively positive effect in the texture of the yoghurt.

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 the present study, we used SPME-GC-MS to identify the VOCs present in fermented milk with different starters. It shows that milk fermented with L.

Amino acid composition analysis
The amino acid content of the fermented milk in different groups was analyzed with the PCA methods. As shown in Figure 4A, L. plantarum A3 fermented yoghurt groups are relatively closer, compared with control group. It can be seen that yoghurt with L. plantarum A3 strain indeed promote the amino acid components formation during the milk fermentation processing, and Ser, Gly and Thr are the main positive amino acids in both PC1 and PC2 during the L. plantarum fermentation procedure ( Figure   4B-4D), which play an important role in the nutrition formation of the fermented milk products.

Fatty acid composition analysis
The  (Table 1).

Electronic tongue analysis
The results of the electronic tongue analysis (

β-galactosidase and lactate dehydrogenase analysis
The activity of β-galactosidase and lactate dehydrogenase in culture media supernatant with different ratio of L. plantarum, L. bulgaricus and S. thermophilus were shown in Figure 7. The activity of β-galactosidase decreased gradually when the proportion of L. plantarum A3 increased, and the activity of lactate dehydrogenase shown similar trend, which the 1:1:1 group had the highest enzyme activity compared with other groups.

Discussion
As a common strain in Sichuan pickle product, L. plantarum A3 also reveals great potential in probiotic yogurt in terms of bacterial population, texture and flavor [12]. Meanwhile, probiotic yogurt has many health benefits and can maintain a sufficient amount in the human gastrointestinal tract, such as milk fermented with probiotic strains such as Bifidobacteria and Lactobacilli [13,14]. Milk fermented with L. plantarum A3 in this research revealed a novel texture characteristic in terms of hardness, acidity and water holding capacity when combined with the traditional L. bulgaricus and S. thermophilus strains.
Most probiotics belong to lactic acid-producing bacteria and exist in forms of yoghurt, fermented pickles or other fermented foods [15]. Probiotics species of Lactobacilli and Bifidobacteria exist in more than 90% of probiotic products and are popular in healthy consumers [16,17]. In this research, L. plantarum has a good lactic acid producing ability, which is essential for the quality of the milk fermentation. In the glucose metabolism pathway, pyruvate is an important intermediate metabolite of glucose, which is dehydrogenated by the action of lactate dehydrogenase to produce lactic acid in lactic acid bacteria. Milk fermented with L. plantarum A3 group have a higher lactate dehydrogenase activity than the control group, and as the proportion of L. plantarum A3 increased, the enzymatic activity of β-galactosidase was also found enhanced. Lactose in milk can be converted to lactic acid during fermentation, and β-galactosidase is capable of hydrolyzing lactose in the most abundant lactose milk to form lactic acid and its byproducts [18,19]. Since strains under symbiotic culture conditions can provide a lower pH environment which may affect the activity of β-galactosidase in the culture media. While, the activity of lactate dehydrogenase in L. plantarum A3 group was higher than the control group, indicating that the reason why acetic acid concentration was higher in L. plantarum A3 groups. Therefore, the strains ratio of 1:1:2 is better for the symbiotic milk fermentation in this research.
In general, probiotic strains are screened based on their safety, nutritional value and health-promoting properties, as well as the fermentation properties that may affect the texture and appearance of the probiotic yogurt [20]. Yoghurt fermentation with L. plantarum A3 indeed has a good quality and nutrition 13 properties compared with the traditional strains. There was a significant difference between the L. plantarum A3 and the L. plantarum A3-free yoghurt on the content of free amino acids depends on the PCA analysis, meanwhile, the main free amino acid components of the B1, B2 and B3 groups, probably due to the proportion of L.
plantarum A3 in the yoghurt. Some studies have shown that the addition of Lactobacillus and Bifidobacteria can increase the concentrations of free amino acids (FAAs) in all probiotic cheeses [11,22]. 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 nonfermented milk [25]. We also found that the with L. plantarum A3 can enhance the level of omega-3 fatty acids profile in the fermented milk, newly discovery revealed that the fatty acid composition involved in the inflammatory response of the cell 14 and n-3 PUFAs have the potential anti-inflammatory effects which may be useful as therapeutic agents in disorders with an inflammatory component [26]. This maybe also the reason why yoghurt consumption can modulate the immune system and prevent the inflammatory process in a mouse model [27].

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Not applicable  Fermentation growth kinetics of strains in symbiotic culture conditions with L. plantarum A3.

Figure 1
Fermentation growth kinetics of strains in symbiotic culture conditions with L. plantarum A3.
21 Figure 2 Texture, water holding capacity and acidity evaluation of the yoghurt fermented with differe 22 Figure 2 Texture, water holding capacity and acidity evaluation of the yoghurt fermented with differe 23 Figure 3 . Volatile flavor profiles of the fermented milk with different proportion of L. plantarum A3. w Electronic tongue analysis of the fermented milk with and without L. plantarum A3 strain. Pri 27 Figure 6 Electronic tongue analysis of the fermented milk with and without L. plantarum A3 strain. Pri The activities of β-galactosidase (A) and lactate dehydrogenase (B) under co-culture conditio Figure 7 The activities of β-galactosidase (A) and lactate dehydrogenase (B) under co-culture conditio