Vol.:(0123456789) European Food Research and Technology (2021) 247:719–736 https://doi.org/10.1007/s00217-020-03660-3 ORIGINAL PAPER Impact of malolactic fermentation with Lactobacillus plantarum on volatile compounds of sea buckthorn juice Niko Markkinen1  · Oskar Laaksonen1  · Baoru Yang1 Received: 18 September 2020 / Revised: 18 November 2020 / Accepted: 22 November 2020 / Published online: 8 February 2021 © The Author(s) 2021 Abstract Malolactic fermentation using sea buckthorn (Hippophaë rhamnoides) juice as raw material was performed with six different strains of Lactobacillus plantarum. Increasing juice pH from 2.7 to 3.5 or adapting cells to low pH (i.e., acclimation) prior to inoculation allowed malolactic fermentation with all tested strains. Moreover, reducing pH of the growth medium from 6 to 4.5 with l-malate had little or no impact on biomass production. Volatile profile of sea buckthorn juice was analyzed with HS-SPME–GC–MS before and after fermentation. A total of 92 volatiles were tentatively identified and semi-quantified from sea buckthorn juice, majority of which were esters with fruity odor descriptors. Esters and terpenes were decreased in both inoculated and control juices during incubation. Microbial activity increased the levels of acetic acid (vinegar like), free fatty acids (cheese like), ketones (buttery like), and alcohols with fruity descriptors. Conversely, aldehydes associated with “green” aroma were decreased as a result of fermentation. Juices fermented with DSM 1055 had the highest acid and alcohol content, while fermentation with DSM 13273 resulted in the highest content of ketones. Compared to inoculation with other strains, fermentation with strains DSM 16365 and DSM 100813 resulted in rapid malolactic fermentation, less production of volatile acids, and lower loss of esters and terpenes important for natural sea buckthorn flavor. Keywords Acclimation · Lactic acid bacteria · Volatiles · GC–MS · SPME · Berry Abbreviations MLF Malolactic fermentation SBJ Sea buckthorn juice GEM General edible medium CAM Cell acclimation medium Introduction Sea buckthorn (Hippophaë L.) is a genus of deciduous shrubs belonging to the family Elaeagnaceae. Eight species have been identified within the genus, originating from dif- ferent regions throughout the Eurasian continent and have been cultivated in Europe, Asia and the North America for commercial purposes [1]. Sea buckthorn produces oval shaped berries of yellow, orange or red color with strong variation both between and within species as well as among cultivars [2]. The berry mesocarp accumulates substantial amount of oil (up to 4% of FW), which consists of triacyl- glycerols, phospholipids, tocopherols, tocotrienols, carot- enoids, and plant sterols. Hydrophilic fraction of the sea buckthorn berry contains high levels of ascorbic acid, fla- vonoids and organic acids [2]. Human trials have associated consumption of sea buckthorn and sea buckthorn products with improved health of mucous membranes [3, 4] and reduction in postprandial insulin response [5]. However, sensory value of sea buckthorn characterized by intensive sourness and astringency presents a great hurdle for utilization of the berry in food industry [6, 7]. Sour taste of sea buckthorn juice has been associated with the high content of organic acids, especially those of malic acid and quinic acid, with the total acid content ranging between 31 and 51 g/L, depending on the variety. Moreover, the juice has low natural sweetness, due to the low total sugar content (19–71 g/L) and low sugar/acid ratio [7, 8]. Astringency of sea buckthorn has been associated with the high content of flavonoids, especially with flavonols and procyanidins [9], * Baoru Yang baoru.yang@utu.fi Niko Markkinen niko.markkinen@utu.fi Oskar Laaksonen oskar.laaksonen@utu.fi 1 Food Chemistry and Food Development, Department of Life Technologies, University of Turku, 20014 Turku, Finland 720 European Food Research and Technology (2021) 247:719–736 1 3 as well as with the total acid content [7]. Recently, ethyl glucose, a β-d-glucopyranose derivative present in the sea buckthorn berry, was found to contribute to bitterness of sea buckthorn [10]. Additionally, the juice of sea buckthorn has high turbidity (Brix 9.3–22.7) [2] due to the presence of insoluble solids and suspended oil droplets [11]. One potential solution to reduce intense sourness of sea buckthorn is malolactic fermentation (MLF), which is used in the wine industry to reduce acidity and to alter aroma in wines. While typically performed using Oenococcus oeni, interest towards Lactobacillus plantarum as malolac- tic starter is increasing due to large cascade of enzyme it produces, potentially altering flavor properties of wines and other food products [12, 13]. Earlier, L. plantarum has been successfully used to improve the aroma profile of mulberry juices [14]. Besides flavor modification, benefits of using L. plantarum for bioprocessing of plant materials include improved shelf-life and food safety [15, 16], increased antioxidant capacity [14, 17, 18], and enhanced nutritional value and probiotic properties [19]. However, the changes in physicochemical properties are both raw material and strain dependent; fermentation of pomegranate juice with L. plan- tarum led to beneficial impact on aroma profile [20], while reducing antioxidant activity [21]. Earlier, MLF has been utilized with O. oeni to reduce acidity and, thus, to potentially affect pleasantness of sea buckthorn [8]. In addition, MLF with L. plantarum has been performed on sea buckthorn juice without pH adjustment or acclimation phase [22, 23]. MLF of sea buckthorn juice led to reduction in total acid content without affecting sugars, subsequently increasing sugar/acid ratio [22, 23]. In addi- tion, flavonols of sea buckthorn were not affected; however, protocatechuic acid content was increased [22]. However, in general, the metabolic activity was limited under these circumstances. Therefore, in this study, our first goal was to determine whether MLF of sea buckthorn can be enhanced by adjust- ing initial pH of the sea buckthorn juice or by preparing L. plantarum starter culture in acclimation medium prior to fer- mentation. In wine industry, acclimation is used to enhance wine malolactic fermentation by inducing stress-related gene expression prior to inoculation through exposure to ethanol, low pH, SO2 and l-malate in a medium rich in nutrients [24]. MLF with L. plantarum has potential to both improve aroma (due to ester, e.g., ethyl lactate, formation) and to produce spoilage off-aromas, such as volatile phenols with animal-like “horse sweat” aromas [25]. On the other hand, β-glucosidase activity can release aroma compounds from non-volatile precursors during fermentation [26]. Therefore, our second goal was to analyze changes in volatile profiles of sea buckthorn juice during MLF with HS-SPME–GC–MS to screen formation of potentially pleasant aromas (e.g., floral esters or alcohols) or fermentation related off-flavors. Due to the previous indication on strain-dependent functional prop- erties of lactic acid bacteria as a result of adaptation to the specific environmental niche [19], six commercially avail- able strains of L. plantarum originally extracted from vari- ous fermented plant-based foods were included in this study. Materials and methods Berry material Frozen sea buckthorn (Hippophaë rhamnoides subsp. mon- golica) berries were purchased from a professional farmer (Vinkkilän luomutuote, Vehmaa, Finland). According to the producer, the berries were a mixture of cultivars ‘Ljubi- telskaja’ and ‘Prozrachnaya’. The berries were frozen right after picking and stored at − 20 °C until use. Juice preparation First, frozen sea buckthorn berries were thawed in a micro- wave at 600 W for 3.5 min. Next, berries were made into a mash with a Bamix immersion blender (ESGE Ltd., Swit- zerland). The juice was extracted from the mash with a fruit press (Chef Titanium XL with AT644 attachment, Kenwood, UK) in batches of ~ 400 g of mash, and the juice was filtered through a cheesecloth to remove solids. Thereafter, juice was pooled, divided into aliquots for each fermentation batch, and stored at − 20 °C until use. Two types of juice were used for fermentation, one with natural pH (2.7) and the other with pH adjusted to 3.5 with 1  M NaOH. Study of malolactic gene of L. plantarum showed that both uptake of l-malate and malolactic fermen- tation rate were highest when extracellular pH was between 4 and 5 [27]. However, as pH is increased, metabolic flux towards fermentation of sugars is increased simultaneously [22], which was undesirable in our work. Therefore, pH 3.5 was selected as a compromise to increase malolactic activity, while limiting conversion of sugars to lactate. Prior to pasteurization, the juices were diluted 1:1 (w/w) and divided into 30 mL aliquots in individual glass vials. The juice samples were pasteurized in a water bath (tem- perature ~ 96 °C) until temperature of the juices reached 90 °C, and this was followed by cooling the juices in an ice bath until 10 °C. Juice temperature was monitored with a thermometer (TM-947SD, Lutron Electronics, South Korea) coupled with a thermocouple probe (Supplementary Fig. S1). After cooling, the pasteurized juice samples were tem- pered for 1 h at + 30 °C in an IF-110Plus incubator (Mem- mert GmbH, Schwabach, Germany), followed by preparation for fermentation. 721European Food Research and Technology (2021) 247:719–736 1 3 Fermentation Preparation of bacterial strains as glycerol stocks Freeze-dried cultures of five strains of Lactobacillus plan- tarum subsp. plantarum (DSM 100813 (originating from grape must), DSM 10492 (olive brine), DSM 1055 (bread dough), DSM 13273 (jojoba meal fermentation), DSM 20174T (pickled cabbage)) and one strain belonging to Lac- tobacillus plantarum subsp. argentoratensis (DSM 16365T, fermented cassava roots) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). The bacterial strains were revived in MRS plates for 48 h at + 30 °C, followed by a transfer of a single colony to 250 mL of general edible medium (GEM), prepared according to a previous report with modifications (dextrose 30 g L−1, soy peptone 20 g L−1, yeast extract 7 g L−1, MgSO4 × 7 H2O 1 g L−1, MnSO4 × H2O 0.05 g L−1, in potassium phosphate buffer 0.01 M, pH 6.3 ± 0.2) [28]. The inoculated GEM was incubated at + 30 °C for 24 h, divided into aliquots and mixed at a ratio of 1:1 with 20% glycerol solution, and stored at − 80 °C until use. Optical density (OD600) linear regression models for estimating cell counts To standardize inoculation rate of the SBJ, optical density (OD600) linear regression models were prepared indi- vidually for each of the used strains. First, a growth curve (measured as change in OD600 over time) for each strain was determined in GEM (Supplementary Fig. S2 and S3), showing an early stationary phase reached after approxi- mately 24 h of fermentation with average cell count of 1–3 × 109 CFU/mL (OD600 = 2.2–2.3). Next, five dilutions were made from the cell culture with pure GEM to reach a linear range of the spectrophotometer (UV/Vis UV3100PC, VWR, PA, USA), corresponding to dilutions 1:30–1:6 and OD600 values between 0.2 and 0.7. Sterile GEM media were used as a blank. Each dilution was enumerated with the via- ble plate count (see Sect. “L. plantarum viability count”) to estimate CFU/mL for each OD600 value. Acclimation medium Cell acclimation medium (CAM) was prepared by adding l-malic acid (4 g/L) to GEM and adjusting pH to 4.5. Cell cultures with CAM was prepared similarly to those per- formed with GEM. Starter culture preparation and fermentation First, a scrape from glycerol stock was revived in a MRS plate for 36–48 h at + 30 °C. Next, a single colony was trans- ferred from the MRS plate to 250 mL of either GEM or CAM followed by incubation at + 30 °C for 24–25 h. Next, 80–90 mL of the bacterial culture was transferred to ster- ile centrifuge tubes; thereafter, the cells were collected by centrifugation (4500 × g, 5 min, RT) and washed twice with PBS (140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). After removal of supernatant, the cells were re-suspended to 5 mL of PBS to produce the starter culture. To estimate the cell count of the starter culture, OD600 was measured from a 1:200 dilution. Finally, 30 mL of pasteurized juice was transferred to autoclaved fermenta- tion vessels, and starter culture was added to juice samples to reach an initial cell count of 8.30 Log CFU/mL juice. In total, four fermentation settings were ran simultaneously for each strain, juices with either initial pH 2.7 or 3.5, inoculated with cells from GEM (pH2.7/GEM and pH3.5/GEM, respec- tively) or CAM (pH2.7/CAM and pH3.5/CAM, respectively). The starter culture cell count was enumerated with viable cell plate count (see Sect. “L. plantarum viability count”). The juice samples were fermented at + 30 °C for 36 or 72 h in an IF-110Plus incubator (Memmert GmbH, Schwa- bach, Germany). Control juices without inoculation with both initial pH 2.7 and 3.5 were incubated simultaneously with the inoculated samples for both 36 and 72 h. All fer- mentations were prepared as triplicates (three parallel inocu- lations). After fermentation, the samples were cooled down in an ice bath, each divided into aliquots and stored at –80 °C until analysis. During the whole experimental procedure, the samples were kept above + 4 °C only when necessary to limit residual enzymatic activity. L. plantarum viability count To estimate viable cell count in cultured media or starter cultures, the cell suspension was first serially diluted (1/10) with PBS, followed by streaking 100 µL of dilution to MRS agar plates (LabM, Heywood, UK) and incubation at + 30 °C for 36–48 h. All plates were prepared in triplicates. Colony counts between 30 and 300 on each plate were considered acceptable for enumeration. Analysis of organic acids The concentrations of l-malate, l-lactate and d-lactate of SBJ before and after fermentation were determined using K-LMAL, K-LATE, K-DATE enzyme kits (Megazyme, Bray, Ireland), respectively. 722 European Food Research and Technology (2021) 247:719–736 1 3 Determination of volatile compounds The volatile compounds in the SBJ samples before and after fermentation were analyzed using a method described ear- lier [29] with modifications. Headspace volatiles from juice sample (2 mL of juice with 10% (w/v) NaCl and 10 µL ISTD (ethyl propionate 100 ppm; nonane 200 ppm)) were col- lected with solid phase microextraction (SPME) with a 2 cm DVB/CAR/PDMS fiber (50/30 μm, Supelco, Bellefonte, PA) at 45 °C for 20 min. Prior to headspace volatile collection, the juice sample was incubated 10 min at 45 °C and the fiber conditioned at 230 °C. Analytical instrument of headspace volatiles consisted of a Trace 1310 gas chromatograph coupled with a TSQ 7000 single quadrupole mass spectrometer (Thermo Fisher Sci- entific, Waltham, MA). The gas chromatograph instrument was equipped with either DB-WAX polar capillary column (60 m × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scien- tific, Folsom, CA) or SPB-624 mid-polarity capillary col- umn (60 m × 0.25 mm i.d. × 1.4 μm film thickness, Supelco, Bellefonte, PA). Within each batch of analysis, the order of the samples was randomized to avoid systematic error from residual enzymatic activity. The temperature program of the gas chromatograph oven was as follows: Tstart 50 °C, hold 3 min; Tend 200 °C, rate 5 °C/min, hold 8 min at 200 °C. For SPB-624 column, addi- tional temperature ramp of Tend 230 °C, rate 10 °C/min, hold 4 min was added to reduce the risk of sample carry-over. The injector temperature was 220 °C and the initial injec- tion mode was splitless; the split valve was opened after 0.10 min from injection. The carrier gas was helium at a flow rate of 1.6 and 1.4 mL/min for DB-WAX and SPB-624 columns, respectively. Mass spectra were detected in elec- tron impact mode at 70 eV with a full scan mode (scan range of 33–300 m/z) and a scan speed 0.2 s. The temperatures of the MS transfer line was 200 °C and 210 °C for DB-WAX and SPB-624 columns, respectively. For both columns, the temperature of the ionization source was 220 °C. Each juice sample (prepared in biological triplicates) was analyzed once; no technical replicates were used. Empty vials and vials with only the internal standards were analyzed with every batch to confirm that no cross contamination occurred between the vials during the analysis. The volatile compounds were identified by comparing mass spectra with standard NIST 08 library, literature data and Kovats retention indices (RI). The RIs of the volatile compounds were calculated based on retention times of C5–C30 alkane mixture (Sigma-Aldrich, St. Louis, MO) determined using the same gas chromatographic condi- tions. Individual volatile compounds were semi-quantified (µg/L) by comparing area of the base peak ion to the area of the base peak ion of ethyl propionate (internal standard) (Table 1), which was selected due to low sample to sample variation in peak area and high number of esters present in sea buckthorn juice. Results gained using DB-WAX column were used for semi-quantification, with a few exceptions (Table 1). Statistical analysis Results are reported as mean ± standard deviation, deter- mined from biological triplicates. Paired Student’s t-test was used to compare pH and independent samples test to compare organic acid concentrations (unequal population size and unequal variances assumed) between untreated and fermented SBJ. For comparison of volatile profiles, Tukey’s test for population with equal variances and one- way ANOVA were performed for multiple comparisons. Differences reaching confidence level of p < 0.05 was con- sidered as statistically significant. For comparison of the content of individual volatile compounds within each strain or juice treatment, statistical analyses were performed with software R 3.2.3 (The R Foundation for Statistical Com- puting, Vienna, Austria) using library agricolae (command HSD.test) [30]. Default parameters of the package was used. To study differences between L. plantarum strains (X = 6, n = 24) and the impact of fermentation time (0 h, n = 12; 36 h, n = 78; 72 h, n = 78), and juice pH and growth media as combined variable (X = 4, n = 36) in relation to the sums of volatile compound subgroups, IBM SPSS 26.0 (SPSS, Chicago, IL, USA) was used. In addition, principal com- ponent analysis (PCA) was carried out using the software Unscrambler X (version 11, Camo Inc., Norway). This was used to illustrate the relationship between volatile composi- tion and the treatments applied to produce fermented SBJ. Results and discussion Production of L. plantarum starter culture As normal basal medium (GEM) has been optimized for growing lactic acid bacteria, it was investigated whether CAM would require higher inoculation level due to reduced or limited growth rate. Earlier work has shown that L. plan- tarum retains moderate to high growth rate even when pH of growth medium is reduced to 4.5 [31]. The growth rate of L. plantarum strains DSM 1055 and DSM 13273 in cell acclimation media (CAM) was meas- ured by following change in optical density (OD600) during incubation. These strains were selected as the former had the lowest and the latter highest viable cell count in GEM after 24 of incubation. Several inoculation levels were tested (single colony, 106–108 CFU/mL) (Supplementary Fig. S3). DSM 13273 showed similar growth rate in CAM as in GEM. However, DSM 1055 showed lower growth rate in CAM than 723European Food Research and Technology (2021) 247:719–736 1 3 Ta bl e 1 Id en tifi ca tio n o f v ol ati le co m po un ds w ith S PM E– GC –M S an d t he ir od or de sc rip to r a nd od or se rie s i n n on -tr ea ted an d f er m en ted se a b uc kt ho rn ju ice No . Co m po un d RI a RI b BP c Fo rm ul a Id en t.d Od or se rie s Od or de sc rip to r Re f.e Re f DB -W AX SP B- 62 4 Ac id s 1  A ce tic ac id * 14 60 14 63 68 4 45 C 2 H 4 O 2 M S, R I Ac id ic Sh ar p, pu ng en t, so ur , v in eg ar 1 2  3- M eth yl bu tan oi c a cid 16 76 17 02 91 7 60 C 5 H 1 0O 2 M S, R I Ch ee sy So ur , s we aty , c he es y, tro pi ca l 1 3  2- Hy dr ox y- 2- m eth yl bu ty ric ac id * 96 7 73 C 5 H 1 0O 3 M S 1 4  H ex an oi c a cid 18 52 18 82 10 55 60 C 6 H 1 2O 2 M S, R I Fa tty So ur , f att y, sw ea t, ch ee se 1 5  H ep tan oi c a cid 19 75 20 00 60 C 7 H 1 4O 2 M S, R I Ch ee sy Ra nc id , s ou r, ch ee sy , s we at 1 6  O cta no ic ac id 20 70 21 09 60 C 8 H 1 6O 2 M S, R I Fa tty Fa tty , w ax y, ra nc id , o ily , v eg eta bl e, ch ee sy 1 7  N on an oi c a cid 60 C 9 H 1 8O 2 M S W ax y W ax y, di rty , c he es y, da iry 1 Al co ho ls 8  E th an ol 93 7 93 8 < 60 0 45 C 2 H 6 O M S, R I Al co ho lic Al co ho lic , e th er ea l, m ed ici na l 1 9  3- M eth yl -1 -b ut an ol 12 11 12 11 78 7 55 C 5 H 1 2O M S, R I Fe rm en ted Fu se l, alc oh ol ic, w hi sk ey , f ru ity , b an an a 1 10  1- He pt an ol 14 61 14 60 56 C 7 H 1 6O M S, R I Gr ee n M us ty, le af y, vi ol et, he rb al, g re en , s we et, w oo dy , p eo ny 1 11 2- Et hy l-1 -h ex an ol 14 92 14 93 57 C 8 H 1 8O M S, R I Ci tru s Ci tru s, fre sh , fl or al, oi ly, sw ee t 1 12  3, 3- Di m eth yl -c yc lo he xa no l 15 78 95 C 8 H 1 6O M S 1 13  B en zy l a lco ho l 18 85 18 92 79 C 7 H 8 O M S, R I Fl or al Fl or al, ro se , p he no lic , b als am ic 1 Al de hy de s 14  A ce tal de hy de 69 0 70 2 < 60 0 44 C 2 H 4 O M S, R I Et he re al Pu ng en t, eth er ea l, ald eh yd ic, fr ui ty 1 15  H ex an al 10 83 10 85 84 6 56 C 6 H 1 2O M S, R I Gr ee n Fr es h, gr ee n, fat ty, al de hy di c, gr as sy , l ea fy , f ru ity , s we aty 1 16  H ep tan al 11 88 11 88 95 0 55 C 7 H 1 4O M S, R I Gr ee n Fr es h, ald eh yd ic, fa tty , g re en , h er ba l, co gn ac , o zo ne 1 17  3- M eth yl -2 -b ut en al 12 12 12 02 84 C 5 H 8 O M S, R I Fr ui ty Sw ee t, fru ity , p un ge nt , b ro wn , n ut ty, al m on d, ch er ry 1 18  O cta na l 12 91 12 92 10 54 41 C 8 H 1 6O M S, R I Al de hy di c Al de hy di c, wa xy , c itr us , o ra ng e, pe el, g re en , f att y 1 19  N on an al 13 96 13 97 11 57 57 C 9 H 1 8O M S, R I Al de hy di c W ax y, ald eh yd ic, ro se , f re sh , o rri s, or an ge , p ee l, fat ty, pe ely 1 20  B en za ld eh yd e 15 29 15 36 10 42 77 C 7 H 6 O M S, R I Fr ui ty Sh ar p, sw ee t, bi tte r, alm on d, ch er ry 1 Al ka ne s 21  4- M eth yl oc tan e 82 3 85 6 86 7 43 C 9 H 2 0 M S, R I Ga so lin e 2 22  N on an e ( IS TD )* * 90 1 90 0 57 C 9 H 2 0 M S, S TD 23  D ec an e 99 9 99 9 43 C 1 0H 22 M S, S TD Ga so lin e 3 24  2, 4- Di m eth yl he pt an e 79 7 80 9 82 5 85 C 9 H 2 0 M S, R I Es ter s 25  E th yl pr op io na te (IS TD )* * 96 1 96 0 74 0 57 C 5 H 1 0O 2 M S, S TD 26  E th yl 2- m eth yl pr op an oa te 96 1 96 9 78 4 43 C 6 H 1 2O 2 M S, R I Fr ui ty Sw ee t, eth er ea l, fru ity , a lco ho lic , f us el, ru m m y 1 27  M eth yl 3- m eth yl bu tan oa te 10 19 10 23 80 4 74 C 6 H 1 2O 2 M S, R I Fr ui ty Ap pl e, fru ity , p in ea pp le 1 28  E th yl bu ty ra te 10 36 10 41 82 8 89 C 6 H 1 2O 2 M S, R I Fr ui ty Fr ui ty, ju icy , f ru it, fr ui ty, pi ne ap pl e, co gn ac 1 29  E th yl 2- m eth yl bu tan oa te 10 52 10 57 87 7 57 C 7 H 1 4O 2 M S, R I Fr ui ty Sh ar p, sw ee t, gr ee n, ap pl e, fru ity 1 30  E th yl 3- m eth yl bu tan oa te 10 72 10 72 88 0 88 C 7 H 1 4O 2 M S, R I Fr ui ty Fr ui ty, sw ee t, ap pl e, pi ne ap pl e, tu tti , f ru tti 1 31  3- M eth yl -1 -b ut yl ac eta te 11 26 11 26 43 C 7 H 1 4O 2 M S, R I Fr ui ty Sw ee t, fru ity , b an an a, so lve nt 1 32  E th yl pe nt an oa te 11 36 11 39 92 7 88 C 7 H 1 4O 2 M S, R I Fr ui ty Sw ee t, fru ity , a pp le, pi ne ap pl e, gr ee n, tro pi ca l 1 33  P ro py l 3 -m eth yl bu tan oa te 11 67 11 59 97 7 85 C 8 H 1 6O 2 M S, R I Fr ui ty Bi tte r, sw ee t, ap pl e, fru ity 1 34  2- M eth yl pr op yl bu tan oa te 11 61 11 63 71 C 8 H 1 6O 2 M S, R I Fr ui ty Sw ee t, fru ity , p in ea pp le, tu tti fr ut ti, ru m m y, ch er ry , a pp le 1 724 European Food Research and Technology (2021) 247:719–736 1 3 Ta bl e 1 (c on tin ue d) No . Co m po un d RI a RI b BP c Fo rm ul a Id en t.d Od or se rie s Od or de sc rip to r Re f.e Re f DB -W AX SP B- 62 4 35  Is ob ut yl 2- m eth yl bu tan oa te 11 79 11 80 85 C 9 H 1 8O 2 M S, R I Fr ui ty Sw ee t, fru ity 1 36  M eth yl he xa no ate 11 85 11 89 95 5 74 C 7 H 1 4O 2 M S, R I Fr ui ty Fr ui ty, pi ne ap pl e, eth er ea l 1 37  Is ob ut yl 3- m eth yl bu tan oa te 11 98 11 93 10 35 85 C 9 H 1 8O 2 M S, R I Fr ui ty Sw ee t, fru ity , a pp le, ra sp be rry , g re en , b an an a 1 38  3- M eth yl bu ty l 2 -m eth yl pr op an oa te 11 83 11 96 10 40 70 C 9 H 1 8O 2 M S, R I Fr ui ty Fr ui ty, w ax y, ap ric ot , p in ea pp le, g re en , b an an a 1 39  E th yl 3- m eth yl -2 -b ut en oa te 12 19 12 31 95 8 83 C 7 H 1 2O 2 M S, R I Fr ui ty, si lag e- lik e 4 40  E th yl he xa no ate 12 33 12 38 10 26 88 C 8 H 1 6O 2 M S, R I Fr ui ty Sw ee t, fru ity , p in ea pp le, w ax y, gr ee n, ba na na 1 41  3- M eth yl bu ty l b ut an oa te 12 56 12 70 10 84 70 C 9 H 1 8O 2 M S, R I Fr ui ty Fr ui ty, g re en , a pr ico t, pe ar, ba na na 1 42  3- M eth yl bu ty l 2 -m eth yl bu tan oa te 12 79 12 82 11 30 70 C 1 0H 20 O 2 M S, R I Fr ui ty Sw ee t, fru ity , c itr us , c he rry , b lu eb er ry , a pp le 1 43  2- M eth yl bu ty l 2 -m eth yl bu tan oa te 12 76 12 84 70 C 1 0H 20 O 2 M S, R I Fr ui ty Sw ee t, fru ity , e ste ry , b er ry , g re en , w ax y, ap pl e 1 44  3- m eth yl bu ty l 3 -m eth yl bu tan oa te 12 96 12 99 11 35 70 C 1 0H 20 O 2 M S, R I Fr ui ty Sw ee t, fru ity , g re en , r ip e, ap pl e, jam m y, tro pi ca l 1 45  P ro py l h ex an oa te 13 24 13 23 11 23 99 C 9 H 1 8O 2 M S, R I Fr ui ty Sw ee t, fru ity , j ui cy , p in ea pp le, g re en , t ro pi ca l 1 46  E th yl he pt an oa te 13 41 13 38 11 26 88 C 9 H 1 8O 2 M S, R I Fr ui ty Fr ui ty, pi ne ap pl e, co gn ac , r um m y, wi ne y 1 47  P en ty l 3 -m eth yl bu tan oa te 13 50 13 51 11 73 85 C 1 0H 20 O 2 M S, R I Fr ui ty Ap pl e f re sh fr ui ty 1 48  2- M eth yl pr op yl he xa no ate 13 53 13 57 11 80 99 C 1 0H 20 O 2 M S, R I Fr ui ty Sw ee t, fru ity , p in ea pp le, g re en , p ea ch , t ro pi ca l 1 49  3- M eth yl bu ty l p en tan oa te 13 46 13 66 11 82 70 C 1 0H 20 O 2 M S, R I Fr ui ty Fr ui ty, ri pe , a pp le, g re en 1 50  3- M eth yl -3 -b ut en -1 -y l 3 -m eth yl bu tan oa te 13 72 13 74 11 50 68 C 1 0H 18 O 2 M S, R I 51  4- M eth yl pe nt yl 3- m eth yl bu tan oa te 14 01 14 02 85 C 1 1H 22 O 2 M S, R I Fr ui ty, pe ac h 5 52  E th yl 3- hy dr ox y- 3- m eth yl bu tan oa te 14 00 14 18 10 08 59 C 7 H 1 4O 3 M S, R I Fr ui ty, g ra ss , g ra in , c as he w 6 53  3- m eth yl bu t-2 -e ny l p en tan oa te 14 21 11 78 68 C 1 0H 18 O 2 M S 54  E th yl 2- hy dr ox y- 3- m eth yl bu tan oa te 14 25 14 33 10 20 73 C 7 H 1 4O 3 M S, R I Fr ui ty Pi ne ap pl e, str aw be rry , t ea , h on ey 1 55  E th yl oc tan oa te 14 37 14 40 12 24 88 C 1 0H 20 O 2 M S, R I W ax y Fr ui ty, w in ey , w ax y, sw ee t, ap ric ot , b an an a, br an dy , p ea r 1 56  H ex yl 3- m eth yl bu tan oa te 14 44 14 50 12 70 85 C 1 1H 22 O 2 M S, R I Fr ui ty Sw ee t, gr ee n, fru ity , a pp le, ap pl e s ki n, str aw be rry 1 57  Is op en ty l h ex an oa te 14 69 14 65 12 80 70 C 1 1H 22 O 2 M S, R I Fr ui ty Fr ui ty, ba na na , a pp le, pi ne ap pl e, gr ee n 1 58  Is op en ty l 3 -m eth yl -2 -b ut en oa te 14 62 14 72 12 18 83 C 1 0H 18 O 2 M S, R I 59  E th yl 4- oc ten oa te 14 83 14 78 12 20 82 C 1 0H 18 O 2 M S, R I Fr ui ty Fr es h p in ea pp le ju icy pe ar 1 60  P en ty l h ex an oa te 15 25 15 16 70 C 1 1H 22 O 2 M S, R I Fr ui ty Sw ee t, gr ee n, fru ity , e ste ry , p in ea pp le, ap pl e, pe ar, fa tty 1 61  P ro py l o cta no ate 15 26 15 25 57 C 1 1H 22 O 2 M S, R I Co co nu t Co co nu t, co co a, co gn ac , w in ey , f att y 1 62  P en t-4 -e ny l h ex an oa te 15 41 12 95 68 C 1 1H 20 O 2 M S 63  2- M eth yl pr op yl oc tan oa te 15 61 15 58 57 C 1 2H 24 O 2 M S, R I Fr ui ty Fr ui ty, g re en , o ily , fl or al 1 64  3- M eth yl bu ty l h ep tan oa te 15 52 15 64 13 78 11 3 C 1 2H 24 O 2 M S, R I He rb al Fr ui ty, he rb al, g ra ss y, ba na na , u nr ip e, ba na na , f ru ity 1 65  M eth yl be nz oa te 16 36 16 37 11 66 10 5 C 8 H 8 O 2 M S, R I Ph en ol ic Ph en ol ic, w in ter gr ee n, alm on d, flo ra l, ca na ng a 1 66  E th yl de ca no ate 16 48 16 45 14 28 88 C 1 2H 24 O 2 M S, R I W ax y Sw ee t, wa xy , f ru ity , a pp le, g ra pe , o ily , b ra nd y 1 67  3- M eth yl bu ty l o cta no ate 16 70 16 66 14 83 70 C 1 3H 26 O 2 M S, R I Fr ui ty Sw ee t, oi ly, fr ui ty, g re en , s oa py , p in ea pp le, co co nu t 1 68  E th yl tr an s-4 -d ec en oa te 16 72 16 74 14 15 11 0 C 1 2H 22 O 2 M S, R I Gr ee n Gr ee n, fru ity , w ax y, co gn ac 1 69  E th yl be nz oa te 16 34 16 81 12 39 10 5 C 9 H 1 0O 2 M S, R I M in ty Fr ui ty, dr y, m us ty, sw ee t, wi nt er gr ee n 1 70  2- Fu ry lm eth yl 3- m eth yl bu tan oa te 16 96 16 96 81 C 1 0H 14 O 3 M S, R I Fr ui ty Be rry , f ru ity , g ra pe , p lu m , r ip e 1 71  M eth yl sa lic yl ate 17 83 17 95 12 0 C 8 H 8 O 3 M S, R I M in ty W in ter gr ee n, m in ty 1 725European Food Research and Technology (2021) 247:719–736 1 3 Ta bl e 1 (c on tin ue d) No . Co m po un d RI a RI b BP c Fo rm ul a Id en t.d Od or se rie s Od or de sc rip to r Re f.e Re f DB -W AX SP B- 62 4 72  E th yl ph en yl eth an oa te 17 85 17 99 13 11 91 C 1 0H 12 O 2 M S, R I Fl or al Sw ee t, flo ra l, ho ne y, ro se , b als am ic, co co a 1 73  E th yl do de ca no ate 18 43 18 51 88 C 1 4H 28 O 2 M S, R I W ax y Sw ee t, wa xy , fl or al, so ap y, cle an 1 74  P he ny lm eth yl 3- m eth yl bu tan oa te 18 94 19 07 14 68 91 C 1 2H 16 O 2 M S, R I Fr ui ty Sw ee t, fru ity , a pp le, pi ne ap pl e, he rb al 1 75  3- M eth yl bu ty l b en zo ate 19 28 19 33 15 14 10 5 C 1 2H 16 O 2 M S, R I Ba lsa m ic Sw ee t, ba lsa m ic, g re en , w ax y 1 76  2- Ph en yl eth yl pe nt an oa te 20 34 20 07 10 4 C 1 3H 18 O 2 M S, R I Fl or al Fr ui ty, ro se , l ea fy 1 77  3- M eth yl bu t-3 -e ny l b en zo ate 19 93 20 14 10 5 C 1 2H 14 O 2 M S, R I 78  P he ny lac eti c a cid is oa m yl es ter 20 16 20 19 43 C 1 3H 18 O 2 M S, R I Ch oc ol ate Sw ee t, ho ne y, co co a, ba lsa m ic, ch oc ol ate , c as to re um , a ni m al 1 Ke to ne s 79  A ce to ne 81 4 81 7 < 60 0 43 C 3 H 6 O M S, R I So lve nt So lve nt , e th er ea l, ap pl e, pe ar 1 80  2- Bu tan on e* 64 3 43 C 4 H 8 O M S Et he re al Ac eto ne , e th er ea l, fru ity , c am ph or eo us 1 81  2- Pe nt an on e* 73 5 43 C 5 H 1 0O M S Fr ui ty Sw ee t, fru ity , e th er ea l, wi ne , b an an a, wo od y 1 82  B ut an e- 2,3 -d io ne 97 7 97 7 86 C 4 H 6 O 2 M S, R I Bu tte ry Bu tte ry , s we et, cr ea m y, pu ng en t, ca ra m ell ic 1 83  2- He pt an on e 11 85 11 86 94 1 58 C 7 H 1 4O M S, R I Ch ee sy Fr ui ty, sp icy , s we et, he rb al, co co nu t, wo od y 1 84  3- hy dr ox yb ut an -2 -o ne 12 87 12 92 78 5 45 C 4 H 8 O 2 M S, R I Bu tte ry Sw ee t, bu tte ry , c re am y, da iry , m ilk y, fat ty 1 85  6- M eth yl -5 -h ep ten -2 -o ne 13 42 13 43 43 C 8 H 1 4O M S, R I Ci tru s Ci tru s, gr ee n, m us ty, le m on gr as s, ap pl e 1 86  2- No na no ne 13 93 13 93 11 47 58 C 9 H 1 8O M S, R I Fr ui ty Fr es h, sw ee t, gr ee n, we ed y, ea rth y, he rb al 1 87  2- Un de ca no ne 15 99 16 06 13 48 58 C 1 1H 22 O M S, R I Fr ui ty W ax y, fru ity , c re am y, fat ty, or ris , fl or al 1 Te rp en es 88  β- Oc im en e 12 52 12 55 10 67 93 C 1 0H 16 M S, R I Fl or al Ci tru s, tro pi ca l, gr ee n, ter pe ni c, wo od y, gr ee n 1 89  P re no l 13 13 13 26 71 C 5 H 1 0O M S, R I Fr ui ty Fr ui ty, g re en , l av en de r 1 90  (Z )-3 -h ex en -1 -o l 13 89 13 89 41 C 6 H 1 2O M S, R I Gr ee n Fr es h, gr ee n, cu t, gr as s, fo lia ge , v eg eta bl e, he rb al, oi ly 1 91  L in alo ol 15 52 15 50 93 C 1 0H 18 O M S, R I Fl or al Ci tru s, flo ra l, sw ee t, bo is, de , r os e, wo od y, gr ee n, bl ue be rry 1 Su lfu r-c on tai ni ng co m po un ds 92  M eth an eth io l 67 5 67 4 47 CH 4S M S, R I Su lfu ro us Ca bb ag e, ga rli c 1 93  D im eth yl di su lfi de 10 78 10 78 94 C 2 H 6 S 2 M S, R I Su lfu ro us Su lfu ro us , v eg eta bl e, ca bb ag e, on io n 1 94  M eth yl pr op yl su lfi de 12 67 61 C 4 H 1 0S M S Al lia ce ou s Al lia ce ou s, cr ea m y, gr ee n, lee k 1 a R ete nt io n in di ce s o f v ol ati le co m po un ds fo r D B- W AX o r s im ila r p ol ar c ol um n in li ter atu re . R efe re nc es : f or c om po un d 70 [5 1] , o th er wi se a cq ui re d fro m N IS T St an da rd R efe re nc e Da tab as e Nu m be r 6 9 b R ete nt io n i nd ice s o f v ol ati le co m po un ds fo r D B- W AX an d S PB -6 24 co lu m ns c B as e p ea k o f m as s s pe ctr um d Id en tifi ca tio n m eth od of vo lat ile co m po un ds . M S, m as s s pe ctr um ; R I, re ten tio n i nd ice s; ST D, au th en tic st an da rd e O do r s er ies an d o do r d es cr ip to r r efe re nc es . 1 . ( ht tp :// ww w. th eg o o ds ce nt sc o m pa ny .co m ); 2. (h ttp :// fla vo rn et. or g/ fla vo rn et. ht m l); 3. M ate ria l s afe ty da ta sh ee t; 4. [5 4] ; 5 . [ 55 ]; 6. [5 6] *S em i-q ua nt ifi ca tio n b as ed on S PB -6 24 ; o th er wi se se m i-q ua nt ifi ca tio n b as ed on D B- W AX ** IS TD in ter na l s tan da rd 726 European Food Research and Technology (2021) 247:719–736 1 3 in GEM, yet reached the target OD600 after 25 h of incuba- tion. Thus, inoculation of CAM with a single colony was confirmed to be adequate. Supplementary Table 1 shows details of the starter cul- tures of each L. plantarum strain used in this research. While the target inoculation level was 2 × 108 CFU/mL, viable plate counts of the starter cultures gave inoculation levels between 1.64 and 2.61 × 108 CFU/mL. While the linear regression models vary between the strains, it should be noted that the model for DSM 1055 underestimated, and the model for DSM 13273 overestimated, the expected cell count. Malolactic fermentation of sea buckthorn juice In MLF, decarboxylation of l-malate produces d- or l-lac- tate, thus increasing pH of the fermented material, unlike the fermentation of sugars by homolactic bacteria, which increases acidity of the food material. Hence, pH of the juice fermented for 36 h increased in correspondence to reduc- tion of l-malate content and to increase of d- and l-lactate (Table 2). More importantly, acclimation of bacterial cells made it possible to ferment SBJ with natural pH with all the studied strains. Here, acclimation medium consisted of normal basal medium for lactic acid bacteria supplemented with l-malate at the concentration of 4 g/L and pH adjusted to 4.5. It has been observed that low pH and presence of l-malate induce expression of mle (malolactic enzyme gene) [12]. Increasing pH of the SBJ to 3.5 prior to fermentation improved MLF to comparable degree as obtained using acclimation medium. Among the studied strains, only strain  DSM 20174 showed production of d- and l-lactate without acclimation in juice with initial pH of 2.7. Comparing results between the two fermentation times (36 h and 72 h) in the samples where MLF was successful, there was no substantial differ- ence in pH, decrease in l-malate content or increase in d- or l-lactate contents. A small decrease in pH occurred after 36 h of fermentation suggesting increase in conversion of sugars into acids. To summarize, with the parameters used here, fermentation time of 36 h was enough for an almost complete malolactic conversion. Compared to other strains, DSM 1055 was an exception, retaining 1.8–3.23 g/L of malate even after 72 h of fermentation. To summarize, acclimation of the L. plantarum strains in a growth media with added l-malate allowed fermenta- tion of SBJ with the natural pH of 2.7 without compromis- ing biomass production during starter culture production. Alternatively, adjusting pH from 2.7 to 3.5 also allowed the successful fermentation of SBJ, without acclimation of the bacteria before fermentation. Volatile profile of initial sea buckthorn juice In total, 91 volatile compounds (Table 2) were identified or tentatively identified from fresh sea buckthorn juice, of which were 53 esters, 7 acids, 6 alcohols, 7 aldehydes, 3 alkenes, 8 ketones, 4 terpenes and 3 sulfur-containing com- pounds. All of the volatile compounds detected from fresh juice were present also in fermented samples in addition to 2-undecanone (compound 87) which was present solely in the fermented samples. The semi-quantification results for individual compounds are presented in Supplementary Table 2. Non-branched, branched, and aromatic esters were detected. Identified non-branched fatty acid esters with varying acyl carbon numbers were, in the descending order of abundance, C6, C8, C3, C10, C5, C7 and C2. The most abundant branched esters were those with acyl group of 3-methylbutanoates, 2-methylbutanoates, 2-methylpro- panoates, or 3-methyl-2-butenoates. Esters of benzoate were the main aromatic esters. The most abundant compounds in the GC–MS chromato- grams were, in the descending order, 3-methylbutyl 3-meth- ylbutanoate, 3-methylbutyl hexanoate and ethyl hexanoate. A majority of the tentatively identified esters have fruity odor descriptor, while esters and terpenes with floral odor description were also detected. Earlier, ethyl and 3-meth- ylbutyl esters with 3-methylbutanoic or hexanoic acids have been found highest in abundance in SBJ. The volatile profile of sea buckthorn berry is dependent on genetic background (i.e., species and cultivar) and growth conditions [32–34]. The main volatile acids detected were acetic acid and medium-chain fatty acids (C6–C9), while fatty acid-derived aldehydes with the same carbon numbers were also detected. Other aldehydes detected were benzaldehyde and acetalde- hyde. Fatty acid-derived ketones with acyl chain lengths of 3, 4, 5, 7, 9, and 11 were detected. However, except for etha- nol and 1-heptanol, no corresponding alcohols to aldehydes or ketones were detected. As sea buckthorn berry accumulates oil in its mesocarp, mostly as triacylglycerols [7, 35], many of the volatile com- pounds detected here, including esters, free fatty acids, alde- hydes and ketones, are derived from metabolism of fatty acids [36]. Non‑microbial impact of incubation time on volatile profile Volatile compounds of food materials are susceptible to alterations due to thermal processing or extended storage [37, 38]. Therefore, it was necessary to separate the effect of incubation on the volatile profile from the impact of micro- bial metabolism during fermentation of SBJ. 727European Food Research and Technology (2021) 247:719–736 1 3 Ta bl e 2 C ha ng e i n p H an d c on ten t o f o rg an ic ac id s ( g/ L) in no n- tre ate d a nd fe rm en ted se a b uc kt ho rn ju ice Va lu e In iti al pH Ti m e ( h) Ju ice M ed ia Fe rm en ted sa m pl es DS M 10 55 DS M 13 27 3 DS M 20 17 4 DS M 10 49 2 DS M 16 36 5 DS M 10 08 13 pH 2.7 0 2.7 0 ± 0. 01 36 2.7 0 ± 0. 01 GE M 2.7 0 ± 0. 01 2.7 1 ± 0. 01 2.7 3 ± 0. 02 * 2.7 1 ± 0. 01 2.7 1 ± 0. 01 2.7 1 ± 0. 01 72 2.7 0 ± 0. 01 GE M 2.7 0 ± 0. 01 2.7 0 ± 0. 01 2.7 4 ± 0. 01 * 2.7 0 ± 0. 01 2.7 0 ± 0. 01 2.7 0 ± 0. 01 36 CA M 2.8 0 ± 0. 01 * 2.8 7 ± 0. 01 * 2.9 5 ± 0. 02 * 2.9 3 ± 0. 01 * 2.9 3 ± 0. 01 * 2.9 4 ± 0. 01 * 72 CA M 2.8 6 ± 0. 02 * 2.9 4 ± 0. 01 * 2.9 8 ± 0. 02 * 2.9 6 ± 0. 01 * 2.9 6 ± 0. 01 * 2.9 7 ± 0. 01 * 3.5 0 3.5 5 ± 0. 01 36 3.5 5 ± 0. 01 GE M 3.6 3 ± 0. 01 3.8 9 ± 0. 01 * 3.9 0 ± 0. 01 * 3.8 7 ± 0. 01 * 3.8 3 ± 0. 01 * 3.8 8 ± 0. 01 * 72 3.5 5 ± 0. 01 GE M 3.6 6 ± 0. 01 3.8 0 ± 0. 01 * 3.8 2 ± 0. 01 * 3.8 0 ± 0. 01 * 3.7 6 ± 0. 01 * 3.8 1 ± 0. 01 * 36 CA M 3.6 9 ± 0. 02 * 3.8 8 ± 0. 01 * 3.8 9 ± 0. 02 * 3.8 9 ± 0. 01 * 3.8 3 ± 0. 02 * 3.8 8 ± 0. 01 * 72 CA M 3.7 5 ± 0. 02 * 3.8 1 ± 0. 01 * 3.8 1 ± 0. 01 * 3.7 9 ± 0. 02 * 3.7 5 ± 0. 01 * 3.8 1 ± 0. 01 * l- m ali c a cid 2.7 0 14 .70 ± 0. 03 36 15 .17 ± 0. 18 GE M 14 .54 ± 0. 58 16 .04 ± 1. 50 11 .76 ± 3. 95 13 .31 ± 0. 66 13 .90 ± 1. 28 14 .14 ± 1. 51 72 14 .70 ± 0. 27 GE M 14 .66 ± 0. 38 14 .66 ± 0. 55 8.9 7 ± 1. 91 * 11 .87 ± 0. 63 14 .72 ± 1. 16 14 .02 ± 0. 59 36 CA M 7.2 9 ± 0. 10 * 1.5 0 ± 0. 30 * 0.0 0 ± 0. 00 * 0.4 6 ± 0. 06 * 1.2 0 ± 0. 74 * 0.3 6 ± 0. 48 * 72 CA M 2.6 7 ± 0. 18 * 0.0 0 ± 0. 00 * 0.0 0 ± 0. 00 * 0.1 1 ± 0. 08 * 0.4 2 ± 0. 12 * 0.2 1 ± 0. 07 * 3.5 0 15 .52 ± 0. 09 36 15 .09 ± 0. 06 GE M 5.3 2 ± 0. 24 * 4.0 1 ± 0. 07 * 1.9 7 ± 0. 17 * 1.6 3 ± 0. 11 * 1.7 4 ± 0. 17 * 2.3 2 ± 0. 12 * 72 14 .22 ± 1. 23 GE M 3.2 3 ± 0. 08 * 0.9 8 ± 0. 02 * 0.0 0 ± 0. 00 * 0.0 0 ± 0. 00 * 0.3 8 ± 0. 06 * 0.0 0 ± 0. 00 * 36 CA M 4.2 7 ± 0. 14 * 0.6 5 ± 0. 05 * 0.0 0 ± 0. 00 * 0.5 5 ± 0. 04 * 0.0 0 ± 0. 00 * 0.0 0 ± 0. 00 * 72 CA M 1.8 0 ± 0. 02 * 0.4 8 ± 0. 03 * 0.0 0 ± 0. 00 * 0.4 7 ± 0. 05 * 0.2 0 ± 0. 05 * 0.0 0 ± 0. 00 * l- lac tic ac id 2.7 0 0.0 0 ± 0. 00 b 36 0.0 0 ± 0. 00 GE M 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 72 0.0 0 ± 0. 00 GE M 0.0 0 ± 0. 00 0.0 0 ± 0. 00 1.4 8 ± 0. 17 * 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 36 CA M 5.6 5 ± 0. 72 * 5.8 0 ± 0. 11 * 8.4 9 ± 0. 59 * 5.6 0 ± 0. 64 * 3.4 9 ± 0. 11 * 5.5 1 ± 0. 61 * 72 CA M 8.1 0 ± 0. 71 * 5.9 8 ± 0. 18 * 5.7 8 ± 0. 00 * 6.4 6 ± 0. 90 * 3.8 6 ± 0. 28 * 4.8 7 ± 0. 61 * 3.5 0 0.0 0 ± 0. 00 36 0.0 0 ± 0. 00 GE M 1.6 9 ± 0. 28 * 4.2 0 ± 0. 32 * 4.8 3 ± 0. 42 * 4.6 5 ± 0. 08 * 2.9 3 ± 0. 51 * 6.9 3 ± 0. 37 * 72 0.0 0 ± 0. 00 GE M 3.1 3 ± 1. 00 * 5.3 8 ± 0. 56 * 5.7 9 ± 0. 86 * 3.7 2 ± 0. 62 * 3.0 0 ± 0. 45 * 6.1 0 ± 0. 24 * 36 CA M 2.7 5 ± 0. 99 * 7.3 9 ± 0. 32 * 5.0 8 ± 0. 41 * 4.6 8 ± 0. 12 * 4.7 4 ± 0. 69 * 5.8 1 ± 0. 56 * 72 CA M 6.8 4 ± 0. 00 * 6.0 8 ± 0. 52 * 5.9 5 ± 0. 57 * 7.3 9 ± 0. 49 * 5.9 1 ± 1. 81 * 5.9 0 ± 0. 35 * 728 European Food Research and Technology (2021) 247:719–736 1 3 To determine how individual volatile compounds and subgroups (volatile acids, esters, terpenes, alcohols, alde- hydes, ketones) were affected by fermentation variables (strain, fermentation time, juice pH, growth media), unsu- pervised classification with principal component analysis was performed for non-fermented juice samples (fermenta- tion time 0 h) in addition to the fermented samples (total n = 168). (Fig. 1). Principal components 1 and 2 together explained 73% of total variance, PC-1 48%, and PC-2 25%. The PCA scores plot (Fig. 1a) shows that PC-1 clearly sepa- rates the non-treated juice from the fermented samples, cor- responding to practically all esters and majority of terpenes (all except linalool) clustering at the left end of PC-1 along with the dummy variable for non-treated juice (“0 h”) in the loadings plot (Fig. 1b). In addition, the content of total esters and total terpenes was decreased significantly (p < 0.001) as incubation time was increased (Fig. 2). Moreover, similar decrease was observed in both inoculated juices and non- inoculated control samples. This suggests that the changes in these esters and terpenes were not related to microbial activity but rather by extended exposure to the fermentation conditions (i.e., incubation temperature). In fruit juices in general, esters are important volatile compounds contribut- ing to the fruity aroma and overall flavor [38], and also in sea buckthorn [39], and thus, limiting loss of the key aroma compounds is important when optimizing the MLF process. Regarding esters, the highest loss of over 50% in normal- ized peak area (0 h vs. 72 h) was observed in ethyl, methyl and propyl esters of butyric acid, 2-methylpropanoic acid, 3-methylbutanoic acid and hexanoic acid (26–34, 36, 39 and 40) (Supplementary Table 2). Similarly, Tiitinen et al. (2006b) observed the reduced content of ethyl 2-methylpro- panoate (26), ethyl 3-methylbutanoate (30) and ethyl hex- anoate (40) in SBJ after MLF with O. oeni. Changes in volatile profile by microbial activity and strain‑dependent differences To investigate in detail the impact of microbial activity on the volatile composition of SBJ, compounds affected by fer- mentation conditions, separated by PC-1 in Fig. 1 (esters and terpenes) were excluded from the second PCA (Fig. 3). In Fig. 3, PC-1 explains 57% of the variance, separating the samples with no or low malolactic activity (on the left) from those with high activities (on the right side). Variables for fermentation times (’36 h’ and ’72 h’) are located close to origo in the loadings plot (Fig. 3b) indicating that fermenta- tion time explains only little variance, as intended, on the first two PCs in the model. However, scores plot (Fig. 3a) shows that different time points are often separated within each strain with samples fermented for 72 h appearing fur- ther to the right side along PC-1 compared to the samples fermented with 36 h. Interestingly, the samples fermented Re su lts a re m ea n ± st an da rd d ev iat io n. Ea ch sa m pl e wa s a na ly ze d at lea st in tr ip lic ate s. Va lu es 0 .00 ± 0. 00 in di ca te co nc en tra tio ns b elo w de tec tio n lim it. A bb re vi ati on s: GE M , g en er al ed ib le m ed iu m ; C AM , c ell ac cli m ati on m ed iu m *S tat ist ica lly si gn ifi ca nt d iff er en ce s b etw ee n un tre ate d (“ Ju ice ” c ol um n, fo r c om pa ris on o f p H on ly T im e = 0  h wa s u se d; fo r c om pa ris on o f o rg an ic ac id co nt en ts, av er ag e o f a ll in cu ba tio n tim es wa s u se d) an d f er m en ted ju ice (p < 0. 05 ) w ith S tu de nt’ s t -te st Ta bl e 2 (c on tin ue d) Va lu e In iti al pH Ti m e ( h) Ju ice M ed ia Fe rm en ted sa m pl es DS M 10 55 DS M 13 27 3 DS M 20 17 4 DS M 10 49 2 DS M 16 36 5 DS M 10 08 13 d- lac tic ac id 2.7 0 0.0 0 ± 0. 00 36 0.0 0 ± 0. 00 GE M 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 72 0.0 0 ± 0. 00 GE M 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.6 6 ± 0. 01 * 0.0 0 ± 0. 00 0.0 0 ± 0. 00 0.0 0 ± 0. 00 36 CA M 2.9 1 ± 0. 14 * 4.9 6 ± 0. 18 * 4.2 5 ± 0. 11 * 4.2 2 ± 0. 31 * 2.7 9 ± 0. 67 * 4.6 2 ± 1. 04 * 72 CA M 3.5 8 ± 0. 19 * 4.5 7 ± 0. 33 * 4.7 3 ± 0. 19 * 5.3 8 ± 0. 11 * 3.7 0 ± 0. 13 * 3.5 8 ± 0. 27 * 3.5 0 0.0 0 ± 0. 00 36 0.0 0 ± 0. 00 GE M 1.7 7 ± 0. 02 * 2.3 7 ± 0. 27 * 2.9 1 ± 0. 22 * 2.4 1 ± 0. 24 * 1.1 4 ± 0. 08 * 2.5 2 ± 0. 07 * 72 0.0 0 ± 0. 00 GE M 2.5 7 ± 0. 05 * 3.3 0 ± 0. 07 * 3.5 3 ± 0. 40 * 3.3 7 ± 0. 14 * 2.4 8 ± 0. 79 * 2.8 0 ± 0. 15 * 36 CA M 2.4 0 ± 0. 10 * 4.0 3 ± 0. 08 * 4.3 6 ± 0. 21 * 2.8 0 ± 0. 04 * 3.5 8 ± 0. 23 * 4.5 6 ± 0. 08 * 72 CA M 5.1 2 ± 0. 52 * 5.4 2 ± 0. 11 * 5.6 5 ± 0. 19 * 3.4 3 ± 0. 32 * 5.8 1 ± 0. 26 * 4.4 7 ± 0. 40 * 729European Food Research and Technology (2021) 247:719–736 1 3 with DSM 20174 (pH 2.7/GEM) separated from other strains with the same fermentation variables. This shows that even modest malolactic activity can produce detectable changes in volatile composition. Total aldehydes, as seen in Fig. 2, were significantly reduced in the fermented samples compared to juice without inoculation. Loadings plot (Fig. 3b) shows that the content of all aldehydes (14–20, green and aldehydic aromas) detected was decreased by fermentation, except 3-methyl-2-butenal Fig. 1 Principal component analysis (PCA) score plots and cor- relation loadings plots based on the data of volatile compounds (99 X-variables) of both non-treated sea buckthorn juice and juice fer- mented with Lactobacillus plantarum (168 samples). The variable numbers in the correlation loadings plots refer to Table  1. Dummy variables are with green font in the loadings plot. Variables written as T(“compound group”) refer to the sum variable for that compound group. Abbreviations: NT, non-treated juice; FC, fermentation control without inoculation 730 European Food Research and Technology (2021) 247:719–736 1 3 (17, fruity aroma) which was increased in abundance. While decrease in aldehydes was universal in all samples with high malolactic conversion, the decrease was less in the juices fermented with DSM 10492 due to the lower reduction of acetaldehyde compared to other strains (Fig. 2, Supplemen- tary Table 2). Previously, fermentation of pineapple, cherry, carrot and tomato juices with L. plantarum caused decrease of almost all detected aldehydes [41]; while, significantly reduced amount of fatty acid-derived aldehydes, namely hexanal, octanal, and nonanal, was observed in rice after fermentation with L. plantarum [36]. In vegetable and fruit juices fermented with L. plantarum, some aldehydes can be reduced to corresponding alcohols [41]. Here, negative correlation between ethanol (8) and acetaldehyde (14), as well as between 1-heptanol (10, green aroma) and heptanal (16) was observed in the fermented juices (Fig. 3b). While both 10 and 16 have green odor descriptor, heptanol has a higher odor threshold [42]. In combination with the overall decrease in aldehydes, changes in aldehyde profile by fer- mentation with L. plantarum could result in the reduction of greenish notes and increase in fruity aroma (17) in SBJ. To highlight the strain-dependent impact on the vola- tile profiles, a third PCA model was created including only samples that were inoculated with L. plantarum at initial pH = 3.5 (Fig. 4). Although the pH2.7/CAM had high malo- lactic fermentation, these were excluded from the model as these samples influenced the model too extensively, as reflected by PC-1 in Fig. 3, where pH2.7/CAM forms a separate cluster from pH3.5/GEM and pH3.5/CAM (effect of pH discussed separately in Sect. “Impact of acclimation and initial juice pH on the volatile profile”). Nonetheless, comparing PCA modeled with only the pH2.7/CAM samples (Supplementary Fig. S4) to Fig. 4 suggests that the strain- dependent differences in volatile profiles appear similar in pH 2.7/CAM, pH 3.5/GEM and pH 3.5/CAM. The alcohol with the highest abundance in fermented samples was 3-methyl-1-butanol (9, fermented aroma) (Sup- plementary Table 2). Due to differences in concentrations of this compound, the juices fermented with strains DSM 1055 and DSM 100813 had elevated volatile alcohol content (p < 0.05), while the lowest content was in samples inocu- lated with DSM 16365 (Fig. 2). In PC-1 and PC-3 load- ings plots (Fig. 4) the two former strains are associated with ethanol (8), 3-methyl-1-butanol (9), and benzyl alcohol (13, floral aroma). This suggests that L. plantarum can introduce potentially both negative fermented aroma (8, 9) and positive floral (13) notes in MLF of sea buckthorn juice. Loadings plot (Fig.  4b) shows that acetic acid (1), 3-methylbutanoic acid (2, cheesy aroma) and medium- chain fatty acids (4–7, fatty and cheesy aromas) are cor- related with total acids. Acetic acid is typically produced through heterofermentive pathways in lactic acid bacteria. While predominantly homofermentive species, genomic studies on L. plantarum have showed the ability to alter- nate between homo- and heterofermentive routes [43]. On the other hand, citrate metabolism (via citrate lyase) into oxaloacetic acid produces acetate as a by-product [12]. In food models, acetic acid has been detected earlier in veg- etable and fruit juices fermented with L. plantarum [41]. Compounds 2 and 4–7 are produced possibly due to increased hydrolysis of the corresponding esters during fer- mentation, possibly due to lipase and/or esterase activity of L. plantarum [44]. Compared to other strains, fermentation with DSM 1055 introduced significantly more volatile acids to SBJ. Significantly lowest levels were in juices fermented with DSM 100813 and DSM 16365, respectively (Fig. 2). As acetic acid has vinegar-like aroma and free fatty acids have been associated with rancid aroma, optimizing fermentation to limit formation of volatile acids is preferable. All identified ketones except 2-butanone (80, ethereal aroma) were positively correlated with samples of high microbial metabolic activity (Fig. 3). 2-Undecanone (87, fruity aroma) was the only compound that was detected solely in fermented samples. 3-Hydroxybutan-2-one (ace- toin, 84) was the most abundant ketone in all fermented samples (Supplementary Table 2). Additionally, loadings plot (Fig. 4b) shows that acetoin and butane-2,3-dione (dia- cetyl, 82) correlated positively with the strain DSM 13273 on PC-2; juices fermented with this strain also had the high- est total ketone content (p < 0.05). Similarly to volatile acids, lowest ketone contents were detected in juices fermented with DSM 100813 and DSM 16365 (Fig. 2). Earlier, the content of 82 and 84 was increased in elderberry juice fer- mented with L. plantarum [45]. In MLF of wines, acetoin and diacetyl are important ketones to enhance buttery and fatty notes. Both acetoin and diacetyl are produced from pyruvate, which in turn originates from either citrate or carbohydrate metabolism [12]. Acetoin can be further con- verted to 2,3-butanediol; however, L. plantarum lacks the enzyme for this conversion (2,3-butanediol hydrogenase) [43], possibly explaining why diacetyl and acetoin, but not 2,3-butanediol, were detected in the fermented juices. While L. plantarum possesses genes to metabolize phe- nolic acids into vinyl derivatives, and further to ethyl deriva- tives [25], no formation of these off-aromas was detected after MLF of sea buckthorn juice. Impact of acclimation and initial juice pH on the volatile profile Exposure of L. plantarum cells to sub-optimal pH and l-malic acid prior inoculation to sea buckthorn (i.e., accli- mation) likely led to activation of genes related to acid stress [12], which in turn allowed MLF of sea buckthorn juice at pH 2.7 (Table 2). However, no significant differences was observed between GEM and CAM juices at pH 3.5 in any of 731European Food Research and Technology (2021) 247:719–736 1 3 Fig. 2 Sums of volatile com- pound subgroups over different fermentation variables. Results are mean ± standard devia- tion. For fermentation control (n = 12) and strains (n = 24), and pH and growth media as combined variable (n = 36) different letter represents groups that are statistically different (p < 0.05). For fermentation time (0 h, n = 12; 36 h, n = 78; 72 h, n = 78) asterisks mark groups that are statistically different (*p < 0.05; **p < 0.01; ***p < 0.001). Tukey’s HSD test of significance was used for comparisons. Y-axis represents semi-quantified volatile content (µg/L) 732 European Food Research and Technology (2021) 247:719–736 1 3 the volatile compound classes (Fig. 2). Additionally, growth mediums explained only little variance in the PCA models (Fig. 4). These together indicate that acclimation in CAM had no secondary effect on the aroma-related metabolism in L. plantarum during the fermentation of sea buckthorn juices. On the other hand, the initial juice pH had significant impact on the observed volatile profiles of the fermented juices. First, the total ester and total terpene content was Fig. 3 Principal component analysis (PCA) score plots and cor- relation loadings plots based on the data of volatile compounds (36 X-variables; terpenes and esters excluded) of sea buckthorn juice fer- mented with Lactobacillus plantarum. Both inoculated and control samples are included (156 samples; excluding samples that have not been incubated). The variable numbers in the correlation loadings plots refer to Table 1. Dummy variables are with green font in load- ings plot. Variables written as T(“compound group”) refer to the sum variable for that compound group 733European Food Research and Technology (2021) 247:719–736 1 3 significantly higher (p < 0.05) in the samples fermented with the initial pH of 3.5 (with high malolactic activity) compared to the pH 2.7/GEM samples with low malolactic activity (Fig. 2). Second, pH 3.5/GEM and pH 3.5/CAM samples had higher total alcohol, acid and ketone content compared to the pH2.7/CAM samples, despite the fact that MLF pro- ceeded efficiently in all these samples (Fig. 2). While the difference was not significant in any of the groups, in Fig. 3 scores plot, pH2.7/CAM samples are clustered into separate group. Here we speculate three possible explanations. First is pH-related matrix effect, as volatile compounds with pH- dependent dissociable group are absorbed in SPME prefer- ably in neutral form, as supported by the previous findings of higher extraction rate of monoterpenols and norisoprenoids from Madeira wines at pH 3.9 compared to pH 2.7 [46, 47]. Second explanation is pH-depentent rate of ester hydrolsis since based on mathematical models, esters are hydrolyzed at a slower rate at a higher pH  [48]. Third explanation is Fig. 4 Principal component analysis (PCA) score plots and cor- relation loadings plots based on the data of volatile compounds (36 X-variables; terpenes and esters excluded) of sea buckthorn juice fermented with Lactobacillus plantarum (72 samples; with inocula- tion and initial pH adjusted to 3.5). Blue and orange colors refer to fermentation time of 36 and 72 h, respectively. A. components 1 and 2; B. components 2 and 3. The variable numbers in the correlation loadings plots refer to Table 1. Dummy variables are with green font in loadings plots. Variables written as T(“compound group”) refer to the sum variable for that compound group 734 European Food Research and Technology (2021) 247:719–736 1 3 reduced or inhibited activity of enzymes related to vola- tile compound formation when extracellular pH is 2.7 [26, 44, 49]. Further research is required to elucidate if the pH- dependent differences in volatile profiles were matrix related or due to enzyme activity (or lack thereof) of L. plantarum. Conclusions We investigated changes in organic acid content and volatile profile of sea buckthorn juice after malolactic fermentation with different strains of Lactobacillus plantarum. Acclima- tion of L. plantarum allowed malolactic fermentation of sea buckthorn juice with its original pH (2.7) with all the stud- ied strains. Increasing juice pH to 3.5 prior to fermentation allowed MLF with the all tested strains regardless the media used for pre-cultivation. Acclimation medium for malolactic fermentation of wines often require high inoculation rates (109 CFU/mL) [50], as the composition of the medium inhibits effective biomass production. In our study, growth rate of L. plantarum in acclimation medium was comparable to growth rate in typical basal medium. While majority of the volatile compounds detected in SBJ were esters of hexanoic and 3-methylbutanoic acid with fruity odor descriptor, a number of alcohols, ketones, alde- hydes, terpenes and acids were also detected. Fermentation time explained most of the variance between the samples, as all of the esters and majority of terpenes were decreased when fermentation time was increased, mostly due to the incubation conditions instead of microbial activities. Micro- bial activities during the fermentation significantly increased the content of volatile acids, ketones, alcohols, while those of aldehydes were decreased. Increase in acid content was due to production of acetic acid by L. plantarum and increased hydrolysis of fatty acid- derived esters. Formation of 3-hydroxybutan-2-one, butane- 2,3-dione and 2-undecanone explained the increase in the ketone content. Fermentation with all the strains reduced content of fatty acid-derived aldehydes. The juices fer- mented with DSM 1055 had significantly more volatile acids and alcohols compared with the samples fermented with other strains, while fermentation with DSM 13273 produced more compounds associated with buttery notes. In contrast, strains DSM 100813 and DSM 16365 produced less volatile acids that contribute to vinegar, fatty and cheesy aromas. In addition, malolactic fermentation proceeded rapidly with these two strains, leading to lower losses of esters and ter- penes important for the original fruity and floral aromas of sea buckthorn. General shortcoming when relating volatile compound analysis to aroma properties is that odor thresholds of vola- tiles vary significantly between compounds and are strongly dependent on sample matrix. Thus, sensory analyses with human subjects are ultimately required to complement the chemical analyses. However, in studies of organoleptic prop- erties, the number of samples need to be kept limited to avoid exhausting the panelists. Therefore, studies screen- ing chemical responses to various fermentation parameters are required. This study provided novel information related to changes in volatile compound profile of sea buckthorn juice in response to acclimation, juice pH, microbial strain and fermentation time. This information can be utilized for development of fermented sea buckthorn products or when designing sensory studies or consumer trials of such products. Supplementary Information The online version contains supplemen- tary material available at https ://doi.org/10.1007/s0021 7-020-03660 -3. Acknowledgements We acknowledge PhD Annelie Damerau for her technical support operating the SPME–GC–MS instrument. Funding Open Access funding provided by University of Turku (UTU) including Turku University Central Hospital. This work was supported by the Doctoral Programme in Molecular Life Sciences (DPMLS) in University of Turku and by personal grants from Raisio Research Foun- dation, Niemi foundation, OLVI foundation, and Magnus Ehrnrooth foundation. Compliance with ethical standards Conflict of interest The authors declare that there is no conflict of in- terest. 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