B. Siegmund & E. Leitner (Eds): Flavour Sci., 2018, Verlag der Technischen Universität Graz 
DOI: 10.3217/978-3-85125-593-5-74, CC BY-NC-ND 4.0 355 
Malolactic fermentation of sea buckthorn (Hippophaë 
rhamnoides L.) berry juice with Lactobacillus plantarum: 
impact on sugars, sugars alcohols, and organic acids 
NIKO MARKKINEN, Sarianna Koivula, Oskar Laaksonen and Baoru Yang 
University of Turku, Food Chemistry and Development, Department of Biochemistry, FI-20014 Turun yliopisto, 
Finland 
Abstract 
Potential to decrease sourness, and thus increase sensory value of sea buckthorn 
berries, by using malolactic fermentation with Lactobacillus plantarum was investigated. 
Sea buckthorn juice samples were fermented with four different L. plantarum strains, and 
chemical changes related to fermentation were determined by analysing sugars, sugar 
alcohols and organic acids as trimethylsilyl-derivates from fermented sea buckthorn 
juices with GC-FID. There was a clear difference in fermentation efficiency between 
studied strains, strain 10492 being the most effective, resulting in total conversion of 
malic acid into lactic acid. Additionally, levels of total sugars maintained comparable to 
the non-treated juice with all strains, and thus sweetness was maintained. Therefore, L. 
plantarum with selected strains is potential candidate for malolactic fermentation of sea 
buckthorn. 
Introduction 
Sea buckthorn (Hippophaë rhamnoides L.) berries contain a versatile combination 
of chemical compounds having health promoting features such water-soluble vitamins 
(C, B1, and B2), fat-soluble vitamins (A, K, and E), fatty acids, flavonoids, and plant 
sterols [1]. However, the sour, bitter and astringent taste characteristics limit its regular 
consumption. The main chemical factors related to the sourness of sea buckthorn are the 
high concentrations of malic and quinic acids. Additionally, strong sourness intensifies 
the perception of astringency [2]. 
One potential treatment to increase the value of sea buckthorn would be to use 
malolactic fermentation, a method currently used for decreasing acidity of sour wines. In 
this process, certain lactic acid bacteria convert malic acid into lactic acid. In the wine 
industry, Oenococcus oeni is the most commonly used lactic acid bacteria [3]. However, 
while being effective in wines, O. oeni has specific nutrient requirements and a relatively 
slow growth rate [4], and thus other candidates for malolactic fermentation of atypical 
materials (such as sea buckthorn) are worth investigating. One potential candidate is 
Lactobacillus plantarum, a bacterial species commonly found in and responsible of the 
fermentation of plant-based lactic acid fermented foods such as sauerkraut and table 
olives. Potential benefit of L. plantarum is in its robustness: it has a relatively fast growth 
rate, tolerance of low pH, and low nutrient requirements [5]. However, studies related 
using L. plantarum as malolactic organism are currently limited. Here we evaluate the 
potential to use Lactobacillus plantarum to decrease acidity of sea buckthorn juice, and 
thus increase its sensory value. 
  
  
Niko Markkinen et al. 356 
Experimental 
Materials 
Frozen sea buckthorn berries (Hippophaë rhamnoides subp. mongolica) were 
obtained from Asterpajutooted OÜ (Tõrva, Estonia). Berries were originated from South 
Estonia, collected from multiple producers by the distributor. Berries were stored at –20 
°C until use. 
Four strains of Lactobacillus plantarum (DSM 16365, DSM 20174, DSM 10492, 
DSM 100813) were obtained from Leibniz Institute DSMZ-German Collection of 
Microorganisms and Cell Cultures (Leibniz, Germany) as freeze-dried cultures. Cultures 
were revived as instructed by the manufacturer, and stored in 10 % (v/v) glycerol in food-
grade medium (FGM) [6] at –20 °C until use. 
Sample preparation and fermentation set-up 
In order to prepare the juice, frozen berries were thawed in a microwave at 650 W 
for 5 minutes with intermittent mixing. Berries were made into a mash with an immersion 
blender. Juice was extracted from the mash with mechanical pressing. Prior to 
pasteurization, the juice was diluted 1:1 with active-carbon filtered water. Juices were 
pasteurized in an autoclave (Systec D-150, Linden, Germany) at 85 °C for 5 minutes. 
After pasteurization, juice samples were cooled down on an ice bath and stored at +4 °C 
for 24 hours before inoculation. 
To produce the starter cultures, each strains was inoculated into 250 ml of FGM by 
a scrape from the glycerol stock with a sterile inoculation loop. Cells were grown at +30 
°C for 24 hours. From each culture, cells were collected with centrifugation at 3410 × g 
for 10 minutes from 200 ml of o/n growth. Cells were washed twice with sterile saline 
solution and concentrated into a volume of 4,5 ml. Each juice sample of 100 ml was 
inoculated with 1 ml of respective cell concentrate. Fermentation was performed for +30 
°C for 72 h in iCinac equipment (Unity Scientific, Milford, USA) equipped with TW8 
water bath (Julabo, Seelbach, Germany). Each fermentation was prepared in duplicates. 
After fermentation, samples were collected in sterile 2 ml tubes and stored at –80 °C until 
analysis. 
Analysis of sugars, sugars alcohols, and organic acids 
First, the juice samples were diluted with reverse-osmosis water to achieve an 
appropriate concentration for the analyses. Aliquots of the diluted samples were dried 
under nitrogen flow, followed by derivatization of the sugars, sugars alcohol and organic 
acids with chlorotrimethylsilane reagent with pyridine and hexamethylsilazane (Tri-Sil 
HTP, Thermo Scientific, Bellefonte, PA, USA). Each sample was prepared in triplicate. 
TMS-derivated samples were analysed by using a Shimadzu 2010Plus gas 
chromatograph (Kyoto, Japan) equipped with flame ionization detector and Shimadzu 
AOC-20i autosampler. Analyses were performed on SPB-1 column (30 m x 0,25 mm ID, 
liquid film 0,25 μm, Supelco, Bellefonte, PA, USA). Internal standards were used for 
quantification, xylitol for sugars and sugar alcohols, and tartaric acid for organic acids. 
External standards were used for the calculation of correction factors and for the 
identification of the analytes. 
  
  
Malolactic fermentation of sea buckthorn (Hippophaë rhamnoides L.) berry juice with Lactobacillus plantarum 357 
Results and discussion 
The purpose of this work was to investigate the potential to utilize malolactic 
fermentation with Lactobacillus plantarum to decrease the acidity in sea buckthorn juice. 
The content of malic acid in the control juice was 11,5 ± 0,08 mg/ml (Figure 1). The level 
of malolactic fermentation varied greatly among the studied L. plantarum strains. Strain 
DSM 10492 was the most effective, with all malic acid converted into lactic acid. Strains 
DSM 20174 and 100813 had moderate conversions, and malic acid contents were reduced 
to 8,73 ± 0,19 and 8,34 ± 0.14 mg/ml, respectively. No conversion was detected with the 
strain 16365. 
 
Figure 1: Lactic acid, malic acid and total sugars (sum of fructose, glucose, myo-inositol and methyl-myo-
inositol) in control sea buckthorn juice and juices fermented with L. plantarum. Error bars present standard 
deviation (N = 6). 
As malic acid is converted into lactic acid, acidity is reduced due to decarboxylation, 
as was observed in the increase in pH of the fermented juices (Figure 2). Although lactic 
acid bacteria can use a variety of carbon sources, including monocarbohydrates such as 
glucose and fructose, the content of total sugars remained similar in the fermented juices 
compared to the control. A similar phenomenon was observed when sea buckthorn juice 
was fermented with other malolactic bacteria, Oenococcus oeni [7]. This is most likely 
due to the high acidity of the material. At low pH, L. plantarum seems to prefer malic 
acid as an energy source over sugars, possibly due to passive diffusion, as the acids are 
predominately present in the protonated form [3]. 
Other identified and quantified compounds were the organic acids citric acid, quinic 
acid, and ascorbic acid, and the sugars ethyl-glucose, myo-inositol, and methyl-myo-
inositol. Additionally, the sugar alcohol L-quebrachitol was identified. Compared to the 
control, samples fermented with strain 10492 had a small but significant (P<0,05) 
decrease in levels of citric acid, fructose, and quinic acid. Comparing the same samples, 
significantly higher levels (P<0,05) of ascorbic acid and ethyl-glucose were measured in 
the fermented samples compared to the control. 
 
0
2
4
6
8
10
12
14
16
Lactic acid Malic acid Total sugar
m
g
/m
l
CONTROL DSM 16365 DSM 20174 DSM 100813 DSM 10492
  
Niko Markkinen et al. 358 
 
Figure 2: Measured pH in sea buckthorn control juice and in juices fermented with L. plantarum. Error bars 
present standard deviation (N = 4). 
All in all, our results indicate that L. plantarum could be used for malolactic 
fermentation of sea buckthorn juice without additional nutrients or by increasing the pH. 
Additionally, other acids, sugars and sugar alcohols remained mostly unfermented. 
Therefore, this method also maintains the sweetness of the berry juice. However, the 
effectiveness of the fermentation is highly affected by the strain. Thus, prior investigation 
of the suitable strains is important. On the other hand, prolonged malolactic fermentation 
can also produce unwanted off-flavours on strain-dependent basis [7], possibly due to the 
production of alcohols [8]. On the other hand, malolactic fermentation with O. oeni was 
shown to increase fruity notes in sea buckthorn juice by releasing more ethyl esters or 
acetate esters of fatty acids [8]. Our work should therefore in the future be combined with 
aroma analysis and sensory evaluations to confirm how the sensory value and the flavour 
of sea buckthorn are affected by lactic acid fermentation with L. plantarum. 
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2,7
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CONTROL DSM 16365
DSM 20174 DSM 100813
DSM 10492