Changes in the Physicochemical Properties and Flavour Compounds of Mulberry after Fermentation with Lactobacillus Plantarum NCU137
1State Key Laboratory of Food Science and Technology of Nanchang University, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China
2School of Food Science & Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China
Received Date: 15/10/2020; Published Date: 26/10/2020
*Corresponding author: Tao Xiong, State Key Laboratory of Food Science & Technology, No. 235 Nanjing East Road, Nanchang, Jiangxi, 330047, PR China
Cite this article: : Zhanggen Liu, Nengneng Su, Zhen Peng, Tao Huang, Shengyang Xiao, Qianqian Guan, Mingyong Xie and Tao Xiong*. Changes in the Physicochemical Properties and Flavour Compounds of Mulberry after Fermentation with Lactobacillus Plantarum NCU137. Op Acc J Bio Sci & Res 5(1)-2020.
The physicochemical properties and volatile flavor compounds in fresh and fermented mulberry by Lactobacillus plantarum NCU137 were detected in this study. The physicochemical property in the initial stage of mulberry fermentation, significantly differed from that in the end of mulberry fermentation. After fermentation, the number of Lactobacillus increased from 107 CFU/mL to 109 CFU/mL in the mulberry fruits, while the sugar contents decreased during the mulberry fruits fermentation. During the fermentation, the organic acids contents increased from 9.51 g/L to 32.64 g/L, the pH value decreased from 3.92 to 3.25, the free amino acids reduced by 36.92%. A total of 54 components were detected in the initial stage of fermentation, among which 33 disintegrated. After fermentation, a total of 38 novel components were found in the mulberries. The aroma components of mulberries significantly changed after fermentation. The alcohols and alkanes content increased from 15.18%, 8.57% to 21.75% and 43.52%, respectively, whereas the percentages of aldehydes decreased from 36.32% to 2.07%.
Keywords: Physicochemical property; Mulberry fruits; Lactobacillus plantarum; Fermentation
The activation of an effective antitumor immune response represents the best way to win cancer. Today, it is known that there are two fundamental immune-mediated anticancer cytotoxic mechanisms, which consist of antigen-independent and antigen-independent cytotoxicity. The antigen-dependent cytotoxicity is mediated by the cytotoxic T lymphocytes (CD8+), while the antigen-independent one is realized by the NK-LAK cell system. The cytotoxic T lymphocytes are mainly activated by IL-12 released from the dendritic cells . On the other side, NK cells are activated by IL-2 secreted by T helper-1 (Th1) lymphocytes , which stimulates NK cells evolution into LAK cells. NK cells play a cytotoxic activity only against laboratory artificial cancer cell lines, whereas LAK cells may destroy fresh human cancer cells drawn from cancer patients themselves . Moreover, Th1 differentiation is promoted by IL-12 itself . Then, IL-2 and IL-12 would represent the two main antitumor cytokines in humans. On the contrary, the antitumor immunity is suppressed by two other major systems, consisting of regulatory T (T reg) lymphocytes  and monocyte-macrophage system . T reg cells exert their immunosuppressive activity on the antitumor immunity by releasing some immunosuppressive cytokines, mainly TGF-beta, IL-10, and IL-35. In more detail, TGF-beta has been proven to suppress the anticancer immunity by inhibiting the secretion of both IL-2 and IL-12 . Macrophages may also inhibit the anticancer immunity through the production of several immunosuppressive cytokines, including IL-6, IL-1beta, TNF-alpha and TGF-beta itself . The secretion of IL-6 is determined by IL-1beta itself. IL-6 has appeared to counteract IL-2-induced transformation of NK into LAK cells, while TNF-alpha may exert a direct lymphocytolytic activity. Moreover, it has been shown that T reg cell generation is promoted by TGF-beta and IL-10 released from some myeloid cell precursors [3-5]. Therefore, the inhibition of TGF-beta secretion and activity could represent a new approach in cancer immunotherapy to enhance the efficacy of the antitumor immunity . Until few years ago, TGF-beta was considered the main endogenous immunosuppressive factor. Moreover, the antitumor efficacy of IL-2 has appeared to be limited by its potential stimulatory role on T reg system and TGF-beta secretion , at least in some experimental conditions. IL-2-induced stimulation of T reg lymphocytes may be abrogated by the concomitant administration of IL-21 . However, more recently another cytokine has been proven to play a fundamental role in the control of cancer growth, the IL-17, which is mainly produced by Th17 lymphocytes , because of its complex influence on the whole cytokine network.
Materials and Methods
Preparation of mulberry samples
Mulberries were provided by Shangzhilvye Berry Co. Ltd. (Heilongjiang, China). Mulberries were washed by distilled water, mixed with high fructose corn syrup (HFSC, w/w, 10%), and beat to puree. The puree was packed into a triangle flask at a sterilization temperature of 90 °C for 20 min. Thereafter, the sterilized puree was cooled. Subsequently, the cooled blueberries was inoculated with the strains NCU137 for fermentation at 37 °C for 72h. The fermented mulberries were collected regularly to analyze the viable cell count of NCU137. Metabolites such as volatile flavor compounds, sugars, organic acids, free-amino acids, and anthocyanins were also detected.
Detection methods of the parameters
Microbiological analysis and determination of pH value: 0.5 g of fermentation samples was dissolved in 4.5 mL of sterile saline (0.85%NaCl, w/v) and diluted to three appropriate gradients. After shaking well, 100µL of the samples was coated on the flat plate of MRS medium. The pH values were determined by a pH meter FE30 (Mettler-Toledo Instruments Company, Shanghai, China). The process was repeated three times for each sample (n=3).
Determination of organic acids and sugars: The mulberry puree was treated following the method by Xiong et al.  Briefly, 1 g of puree samples was diluted by adding 2.5 mL of distilled water into the 5 mL centrifuge tubes. The mixture was centrifuged at 1200 rpm for 15 min by using a high-speed micro-centrifuge (Hunan Xiangyi Labs, Hunan, China). Then, the supernatant was further filtered through the membrane filter (pore diameter, 0.22 μm). The standard curves of organic acids and sugars were established by Agilent 1260 HPLC (Agilent Technologies, Inc., Santa Clara, USA). Organic acids and sugars were separated by the Aminex® HPX-87H Ion Exclusion Column (300 mm.× 7.8 mm, 20 µm particle size, Catalog 125-0140) with sulfuric acid (0.6 mM) as the mobile phase at 45 °C. Refractive index detector and ultraviolet detector (210 nm) were used for the detection of sugars and organic acids, respectively. Thereafter, 20µL of each sample was injected into loading valve and run 25 min at 0.5 mL/min flow of the mobile phase.
Determination of free ammo acids: Free amino acids were analyzed according to the method by Wan et al.  with slight modification. Briefly, 8 g of sample was centrifuged at 3000 rpm for 5 min, and 1 mL of supernatant was added into 9 mL of sulfosalicylic acid (2%, w/v). After standing for 15 min, the mixtures were centrifuged at 3000 rpm for 20 min and filtered through the 0.22 µm membrane. Processed samples were loaded on a model S433D automatic amino acid analyzer (Sykam Corp, Munich, Germany) for amino acids analysis.
Determination of volatile constituents: Samples were treated for determination of volatile constituents following the method by Liu et al.  with slight modification. Briefly, 1g of NaCl was added to 5g of sample and stirred for 1 min, and then packed into a 15mL the solid-phase micro-extraction (SPME) bottle fitted with a polite trafluoroethylene/silicone septum/aluminum cap. After the bottle was heated at 45 °C for 30 min in water bath, SPME fiber was inserted to the bottle for another 30 min. The processed samples were analyzed by headspace solid-phase microextraction (HS-SPME) coupled with GC-MS (Agilent Technologies, Inc., Burwood, Australia). Parameters were set as follows: an SPME fiber, 50/30 mm DVB/Carboxen™/PDMS Stable Flex™, was mounted in the manual SPME holder. By insertion through the septum of the sample bottle, the fiber was exposed to the sample headspace prior to the desorption of the volatiles at 250 °C for 5 min into the splitless injection port of the GC-MS equipped with a 5973-mass selective detector and using an HP-5MS capillary column (30 m 0.25 mm I.D. and 0.25 mm film thickness). In addition, helium was used as the carrier gas. Programmed temperature elution was employed with an initial temperature of 40 °C for 5 min, which was then ramped to 240 °C at 10 °C/min and held at 240 °C for 2 min. Electron impact ionization was performed using an electron energy of 70 eV and a mass range of 20–350 U. The components were identified by comparison of their relative retention times and mass spectra with the standards in the Wiley7n.1 library data of the GC-MS system.
The data such as the sugars, organic acids, pH value and microbiological growth curve were plotted using representative data from 12 fermentation stages. The error bars represent the standard deviation of three independent measurements (n=3). The graphs were drawn by SigmaPolt 12.5. The data were statistically analyzed using ANOVA (SPSS software, IBM Corporation, Armonk, New York, USA) for determination of statistically significant difference between the different values at 95% confidence interval.
Results and Discussion
The cell count and pH value changes
As shown in Figure 1, the initial number of the Lactobacillus plantarum NCU137 was 1.6×107 CFU/mL, after inoculation, and remained at the inoculation level (1.6×107 CFU/mL) at the 4th hour fermentation, which could be related to the lactobacillus bacteria was adapted to the fermentative environment. During 4-32 h, and the NCU137 number increased from 1.6×107 CFU/mL to 3.91×109 CFU/mL and reached a peak at 32 h (3.91×109 CFU/mL). After 40h, the Lactobacillus plantarum NCU137 number sharply dropped to 1.31×109 CFU/mL, and then maintained this level until the end of the fermentation. The pH value decreased from during 0-20h, from initial 3.92 to 3.47 at the 20th hour, and then dropped slowly to 3.25 at the end of the fermentation.
Figure 1: Viable cell counts of NCU137 and change in pH value during mulberry fermentation.
Sugar and organic acid changes
Fructose, glucose, and sucrose are the major sugar components in fruits juice and could be utilized by LAB [20-22]. As shown in Figure 2, the initial contents of sucrose, glucose, and fructose decreased from 4.67, 65.91, and 63.09 mg/mL to 3.02, 31.51, and 26.45 mg/mL at the end of fermentation, respectively, indicating the glucose and fructose were the major sugars utilized by NCU137 during fermentation. These findings were similar to the previous reports that glucose and fructose were the dominant sugar metabolized by Lactobacillus in fruits juice and an increase in glucose and fructose during the fermentation of juice by Lactobacillus . Compared to the glucose, fructose was consumed more slowly during fermentation, which matched the result of cabbage fermentation by Xiong. During the 0-8h, the consumption of sugars (sucrose, glucose, and fructose) were relatively slow, which may be related to the lag period of lactic acid bacteria NCU137. After 8 h, the contents of (sucrose, glucose, and fructose) sharply decreased, and the NCU137 were in the logarithmic growth period as mentioned 3.1, leading to a large consumption of sugar.
Figure 2: Change of sucrose, glucose, and fructose during the fermentation of mulberry by NCU137.
As shown in Figure 3, the initial contents of citric acid, oxalic acid, succinic acid, and isobutyric acid were 4.41, 2.31, 0.55, and 2.24 g/L, respectively. In general, oxalic acid and citric acid are recognized as the principal organic acid in Mulberry [24,25]. After fermentation, the change of oxalic acid was relatively small, while the content of citric acid increased to 7.07 g/L, which was different that a decrease in citric acid is observed during the mulberry fermentation and other fruits juice. We speculate that a large amount of citric acid was released from the juice during fermentation. Although the partial catabolism of succinic acid by LAB remains unclear in fruits juice fermentation, the lactobacillus strains used in fermented juice and vegetables caused losses of succinic acid . As the main organic acids of the end of fermentation, the lactic acid contents increased from 0 g/L to 19.71 g/L, which enhances the microbial stability and confers a pleasant taste to the fermented food products [26,27].These results indicated that fermentation not only changed the content of organic acids of mulberry but also enriched the kinds of organic acids of mulberry.
Figure 3: Change of organic acids during the fermentation of mulberry by NCU137.
Changes of free amino acids after fermentation by the Lactobacillus plantarum NCU137
Sixteen amino acids were observed in this study. These amino acids included aspartic acid (Asp), serine (Ser), lysine (Lys), glycine (Gly), alanine (Ala), cysteine (Cys), valine (Val), methionine (Met), Isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), glutamate (Glu), arginine (Arg), and proline (Pro).As shown in Table 1, the total concentration of free ammo acids (FAA) in the initial stage of fermentation was approximately 1.0578 mg/mL and then decreased to about 0.667, which reduced by 0.3908 mg/mL after fermentation. It was noted that the serine is the most abundant amino acid and decreased from 53.54% to 34.00% of the original total free ammo acids, which could be due to the conversion of serine to pyruvate via serial reactions by deamination [28-30]. In total, a significant decrease (P＜0.05) in the Asp, Ser, Ala, Val, Ile, Leu, Tyr, Phe, Arg, Pro was found after fermentation, while a significant increase (P＜0.05) in the Gly, Cys, Met and Glu was found after fermentation. According to the taste characteristics, the amino acids are classed into bitter amino acids (histidine, arginine, leucine, lysine, valine, phenylalanine, and Isoleucine), sweet amino acids (glycine, alanine, proline, serine, threonine, and methionine), umami amino acids (aspartic acid and glutamate), and astringent amino acid (tyrosine) [31,32]. It total, the contents of bitter amino acids, sweet amino acids, umami amino acids, and astringent amino acid decreased after fermentation. In particular, the content of sweet amino acids decreased the most. Therefore, after fermentation, the sweetness, bitterness and freshness of blueberry decreased, especially the sweetness.
Table 1: Change of free ammo acids after fermentation of mulberry.
**correlation is significant at the 0.01 level, *correlation is significant at the 0.05 level.
Changes of volatile constituents after fermentation by the Lactobacillus plantarum NCU137
The volatile constituents of mulberry were detected by GC-MS (Figure 4). Compared with the volatile flavors of the initial stage of fermentation (Figure 4B), some new volatile flavors of the end of fermentation stage appeared at the acquisition time of 10-16 min (Figure 4A). Based on the variation of functional group, the volatile constituents were classified into nine parts (alcohols, acids, esters, aldehydes, ketones, phenols, alkanes, alkenes, and others) (Table 2). 54 and 59 kinds of volatile compounds were detected in the initial stage of mulberry fermentation and the end of fermentation stage, respectively. In total, a total of 92 kinds of volatile substances were detected in this study, and 21 kinds of volatile substances were both found At the beginning and end of mulberry fermentation, while 33 kinds of volatile substances were unique at the beginning of mulberry fermentation and 38 kinds of novel substances were unique at the end of mulberry fermentation, which indicated that fermentation process affects the flavor composition in mulberry. Alcohols (15.18%), aldehydes (36.32%), and others (15.00%) were the major volatile at the beginning of mulberry fermentation. Other components, such as acids, esters, ketones, phenols, alkanes, and alkenes were approximately 1.74%, 4.47%, 7.04%, 5.85%, 5.83%, and 8.57%, respectively. After fermentation, the contents of alcohols, acids, esters, phenols and alkenes increased from 15.18%, 1.74%, 4.47% and 5.85%, 8.57% to 21.75%, 4.65%, 5.36%, 8.22% and 43.52%, respectively, while the contents of aldehydes, ketones and alkanes decreased from 36.32%,7.04% and5.83% to 2.07%,1.15% and 3.99%, respectively. Previous study reported that the number of alcohols and esters increased during fermentation, and improvement of alcohol content may potentially further enhance the fragrance of fermented juices , which indicated the aroma of mulberry could be improved after fermentation. Researchers have reported that the microorganisms can convert some aldehydes into alcohols . Moreover, the increase of alcohol and decrease of aldehyde in fruit juice after fermentation are also reported in other studies . In total, after fermentation, the variety of volatile constituents in mulberry were greatly enriched and the content of total volatile constituents in mulberry also increased, indicating that fermentation plays an important role in improving the flavor of mulberry.
Figure 4: GC-MS total ion chromatogram of volatile compound profiles observed at 0 h and 48h.
Table 2: Change of volatile constituents after fermentation of mulberry.
We analyzed the dynamic change of components for mulberry fermentation using the Lactobacillus plantarum NCU137 as the fermentative strain. In total, after fermentation, the Lactobacillus counts, organic acids contents, alcohols and alkanes content contents significantly increased, while the pH value and contents of sugar, free amino acids and aldehydes significantly decreased. In addition, the changes of non-volatile flavor and volatile flavor compounds in mulberry would improve the sensory properties and flavor of product significantly. Lactic acid fermentation can be used as a preservation technique for the processing of mulberry juice with enhanced phytochemical, volatile and sensory qualities. Furthermore, processing mulberry into fruit juice makes good use of mulberry, which could be used as a basis for studying the economic viability of producing fermented mulberry derivatives. Thus, fermented mulberry juice exhibits a huge development space and market potential. Further studies should be focused on studying the correlation between Lactobacillus plantarum NCU137 and the specific flavor using omics analysis techniques.
This work was supported the National Key Research and Development Program of China(2017YFD0400503-3), National Natural Science Foundation of China (No.31760457), Construction of Innovation Team in Science and Technology of Jiangxi Province (Project N0.20181BCB24002), and State Key Laboratory of Food Science and Technology Nanchang University (Project No. SKLF-ZZB-201912) are gratefully acknowledged.
Zhanggen Liu and Nengneng Su contributed equally to the article
Declaration of Interest
The authors declare that they have no conflict of interest.
1. Aramwit P, Bang N, Srichana T (2010) The properties and stability of anthocyanins in mulberry fruits. Food Research International 43: 1093-1097.
3. Ercisli S, Orhan E (2007) Chemical composition of white (Morus alba), red (Morus rubra) and black (Morus nigra) mulberry fruits. Food Chemistry 103: 1380-1384.
4. Pérez-Gregorio MR, Regueiro J, Alonso-González E, Pastrana-Castro LM, Simal-Gándara J (2011) Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.). LWT - Food Science and Technology 44: 1793-1801.
6. Liang L (2012) Chemical composition, nutritional value, and antioxidant activities of eight mulberry cultivars from China. Pharmacognosy magazine 8: 215.
7. Chen C, You LJ, Abbasi AM, Fu X, Liu RH (2015) Optimization for ultrasound extraction of polysaccharides from mulberry fruits with antioxidant and hyperglycemic activity in vitro. Carbohydr Polym 130: 122-132.
8. Chen C, Zhang B, Fu X, Liu RH (2016) A novel polysaccharide isolated from mulberry fruits (Murus alba L.) and its selenide derivative: structural characterization and biological activities. Food & Function 7: 2886-2897.
12. Mena P, Sánchez-Salcedo EM, Tassotti M, Martínez JJ, Hernández F, et al. (2016) Phytochemical evaluation of eight white (Morus alba L.) and black (Morus nigra L.) mulberry clones grown in Spain based on UHPLC-ESI-MSn metabolomic profiles. Food Research International 89: 1116-1122.
14. Hashemi SMB, Jafarpour D (2020) Fermentation of bergamot juice with Lactobacillus plantarum strains in pure and mixed fermentations: Chemical composition, antioxidant activity and sensorial properties. Lwt-Food Science and Technology pp. 131.
16. Xu X, Bao Y, Wu B, Lao F, Hu X, et al. (2019) Chemical analysis and flavor properties of blended orange, carrot, apple and Chinese jujube juice fermented by selenium-enriched probiotics. Food Chem 289: 250-258.
17. Kwaw E (2018) Impact of ultrasonication and pulsed light treatments on phenolics concentration and antioxidant activities of lactic-acid-fermented mulberry juice. Lwt-Food Science and Technology 92: 61-66.
21. Hashemi SMB, Khaneghah AM, Barba FJ, Nemati Z, Shokofti SS, et al. (2017) Fermented sweet lemon juice (Citrus limetta) using Lactobacillus plantarum LS5: Chemical composition, antioxidant and antibacterial activities. Journal of Functional Foods 38: 409-414.
22. Xu X, Bi S, Lao F, Chen F, Liao X, et al. (2020) Comprehensive investigation on volatile and non-volatile metabolites in broccoli juices fermented by animal- and plant-derived Pediococcus pentosaceus. Food Chem 341: 128118.
24. Zhang Q (2020) Characterization of gamma-aminobutyric acid (GABA)-producing Saccharomyces cerevisiae and coculture with Lactobacillus plantarum for mulberry beverage brewing. J Biosci Bioeng 129: 447-453.
26. Multari S (2020) Effects of Lactobacillus spp. on the phytochemical composition of juices from two varieties of Citrus sinensis L. Osbeck: 'Tarocco' and 'Washington navel'. Lwt-Food Science and Technology pp. 125.
27. Peng W, Meng D, Yue T, Wang Z, Gao Z (2020) Effect of the apple cultivar on cloudy apple juice fermented by a mixture of Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus fermentum. Food Chem 340: 127922.
29. Ardo Y (2006) Flavour formation by amino acid catabolism. Biotechnol Adv 24: 238-242.
32. Liang ZC, Su H, Lin XZ, He ZG, Li WX, et al. (2020) Microbial communities and amino acids during the fermentation of Wuyi Hong Qu Huangjiu. Lwt-Food Science and Technology pp. 130.
33. Takase H, Sasaki K, Kiyomichi D, Kobayashi H, Matsuo H, et al. (2018) Impact of Lactobacillus plantarum on thiol precursor biotransformation leading to production of 3-sulfanylhexan-1-ol. Food Chem 259: 99-104.