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Assessment of Process Variables on Vitamin B12 Production in Fermented Dairy

Product Including Propionic Acid

Article  in  Current Nutrition & Food Science · December 2018

DOI: 10.2174/1573401315666181204105601

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Current Nutrition and Food Science, 2018, Volume 1

Assessment of Process Variables on Vitamin B12 Production in Fermented

Dairy Product Including Propionic Acid

Ramona Massoud1, Kianoush Khosravi-Darani2*

1
Department of Food Research, Standards Organization, Tehran, Iran
2
Research Department of Food Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition
Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, P.O. Box: 19395-4741, Tehran, Iran

Abstract: The fermented dairy products which produced by various microorganism’s activity provide valuable nutrients
for human. Fermentation affects the physicochemical and organoleptic characteristics of foods as well as human health. In
the present review, we describe the production of vitamin B12 in a fermented dairy product by Propionibacterium species.
The effect of the process variables on vitamin B12 production in fermented dairy products e.g. pH, temperature, different
carbon and nitrogen sources as well as type and size of inoculum, the fermentation time and fermentation strategy, etc. are
discussed. Finally, fermentation strategy, inoculum preparation of Propionibacterium freudenreichii subsp. Shermanii,
methods of determining biomass and Vitamin B12 concentration are reviewed and their effects on vitamin B12 production
are mentioned. Propionibacteria are so popular due to their unique characteristics such as being safe, convenient and
economical. Among all the highest efficiency was observed by P. freudenreichii. To achieve this purpose some
appropriate situations, need to be considered. The best carbon source for this bacterium to produce biomass is lactate, the
suitable pH for growth of the Propionibacterium species is in the range of 6 to 7 and the fed-batch is most preferable for
vitamin B12 production.

Keywords: Fermentation, Dairy, Vitamin B12, Carbon, nitrogen, Propionic acid.

1. INTRODUCTION The fermented dairy products with various appearances,

flavors and textures are dependent to their microbial
Vitamin B12 plays key role in human health due to its
metabolism. The metabolites and enzymes are provided by
contribution in fatty acid synthesis and energy production different microorganisms [7,8]. Fermented dairy products are
[1]. Since chemical production of vitamin B12 is so
so desirable due to their better sensory properties, longer
complicated (including 70 pathway), it is not a preferred
shelf life [9,10]. Anyway the research is continuing to
method for scale up production [2]. So, biosynthesis of minimize total cost of fermentation by using new healthy
vitamin B12 are more interesting which is usually to
and cheap substrates and also optimization of production
fermentation process by Propionibacterium freudenreichii
condition. Recently the application of milk as substrate for
ssp. shermanii and Pseudomonas denitrificans [3]. P. production of a new fermented beverage containing vitamin
freudenreichii ssp. shermanii is preferred, because it has
B12 as well as propionic acid has been introduced [11,12].
obtained the GRAS (generally recognized as safe) status
Due to interesting properties of propionic acid as satiety
from the United States, Food and Drug Administration inducer the fermented drinks contains various health
(FDA) [4]. Fermentation is the oldest method for food
beneficial ingredients including lactic, acetic and citric acid
preservation since many years ago and different from the
as well as Vit B12 [13].
undesirable growth of microorganisms in food and the
microbial spoilage [4,5]. The variety of the fermented 1.1. Fermentative microorganisms in Milk
products is directly dependent on in milk, microbial species
and their metabolic products [6]. Molds, yeasts, and bacteria, Lactic acid bacteria and
Propionibacterium are the most common microorganisms
used in fermented products and molds are used in less
*Address correspondence to this author at the Department of Food quantity in food fermentation. There are many
Technology, National Nutrition and Food Technology Research Institute,, microorganisms used to produce fermented products (Table
Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti
University of Medical Sciences, P.O. Box: 19395-4741, Tehran, Iran;
1) [14].
Tel: +98-21-22086348; Fax: +98-21-22376473;
E-mails: kiankh@yahoo.com; k.khosravi@sbmu.ac.ir

XXX-XXX/14 $58.00+.00 © 2014 Bentham Science Publishers

2 Current Nutrition and Food Science, 2018, Vol. 0, No. 0
Table 1. Microorganisms producing fermented products
Bacteria genera species
Lactobacillus L. delbrueckii lactis, bulgaricus, acidophilus,
helveticus, casei, brevis, fermentum, kefir
Leuconostoc L. mesenteroides dextranicum, cremoris, lactis
Pediococcus P. pentosaceus, acidilactici
Propionibacterium P. freudenreichii shermani, freudenreichii
Streptococcus S. lactis diacetylactis, cremori, thermophilus
Bifidobacterium B. bifidum, longum, infantis, breve, aceti
Acetobacter A. aceti
Yeasts Torulaspora delbrueckii, Kluyveromyces
marxianus subsp. marxianus, Kluyveromyces
marxianus subsp. Bulgaricus, Candida kefir
Saccharomyces cerevisiae
1.2. Fermented milk
The low pH in fermented products and the antimicrobial
compounds produced by bacteria inhibit the growth of
undesirable microorganisms, so fermented milk are more
stable against spoilage. Yeasts and molds may cause
problems in these products which can be controlled by
having good hygiene practices during fermentation and
production [15]. Some factors affecting the antimicrobial
activity of bacteria are the low pH of product, organic acid
such as propionic, lactic, acetic and formic acid in product,
bacteriocins, diacetyl, ethanol, reduction of nutrients and the
redox potential. Each of these factors, can prevent the growth
of undesirable bacteria in the product [16]. Fermented dairy
products as the carriers of probiotic bacteria, have noticeable
nutritional and technological characteristics. Milk is a
suitable environment for probiotics growth and activity as
well as its valuable properties for human [10, 17]. Using
propionibacterium as the microorganisms to produce
fermented products have the advantage of producing vitamin
B12 during the growth and also increasing the nutritional
value of the products [18,19].
1.3. The production of fermented products by
Propionibacterium species
Propionibacteria as well as other probiotics have been known
as gram-positive, catalase-positive, immobile, non-spore
forming, optional anaerobic and rodshaped bacteria. After
fermentation, 3 moles of lactic acid changes to 2 mole of
propionic acid as the main product, and 1 mole of acetic acid
and 1 mole of carbon dioxide [20, 21]. Propionic acid is a
clear, colorless liquid with a slightly sweetish odor and
soluble in water and alcohol. It is also called Ethane
carboxylic acid with chemical formula C3H6O2. Propionic
acid and its sodium and calcium salts have been used as
mold and bacteria inhibitors and also a preservative in a wide
variety of food such as bread and cereals, processed meat,
whey and other dairy products [22, 23]. This acid can be
released and transferred through bacterial membrane to the
cytoplasm due to the hydrophobic properties, then
breakdown to a proton and anion propionate inside the cell
that results in intracellular leakage of proton. In order to
maintain the functional gradient of the protons through the
cell membrane, more ATP should be consumed to remove
Khosravi et al.
proton out of cell which leads to the reduction of available
ATP for the cell metabolism and thereby prevents the fungal
and bacterial growth [24, 25]. Many studies have been done
on the use of the Propionibacterium species as a starter in
cheese making and only in the study of Ruijschop et al.,
(2008), Pseudomonas freudenreichii was used in making
fermented dairy beverage. In this study, the satiety effect of
fermented dairy beverage contains propionic acid was
investigated. Results showed that participants felt
significantly fuller and had less desire to eat after
consumption of the fermented dairy beverage. This result
confirmed that propionate is the active component in satiety
[13, 26].
2. The effect of the process variables
2.1. pH and temperature
Controlling the temperature and pH are the two important
factors in increasing the efficiency of fermentation process.
The results of studies indicate that the optimum pH for
fermentation is about 7 and less than pH = 4, the cell growth
would stop [27, 28]. Other results showed that the suitable
pH for growth of the Propionibacterium species is in the
range of 6 to 7. The maximum pH for growth is 8.5 and the
minimum is 4.6 and in less than 4.6 more volume of
inoculum is required [29]. P. freudenreichii can be viable in
digestive system and would survive at PH 3 to 4. The bile
salts of 0.5% concentration, does not affect the viability of P.
freudenreichii [30]. These bacteria grow in the temperature
of 7 to 45 °C [31]. Propionibacterium species are able to
grow in the absence or low concentrations of oxygen [32].
It is observed that aeration can reduces the production of
propionic acid from P. freudenreichii subsp. Shermanii,
while the cell mass increases. The lower concentrations of
propionic acid under aerobic conditions could be due to the
high density of cells in these conditions [33, 34]. In addition,
the advantage of dairy Propionibacterium is that it can be
produce vitamin B12 without any additional processes [35,
36]. The nutrients affecting the vitamin B12 production are
the amino acids and minerals such as Cobalt in the medium
[37]. In nature, some of bacteria and archaea are able to
produce this vitamin by the 30 enzymes in aerobic and
anaerobic ways [38]. Both aerobic and anaerobic methods
consist of four levels of ALA formation, CORIN ring
formation, the axial ligand formation and the Cobalamin
formation [39, 40].
Recently, a semi-continuous method is applied to produce
this vitamin by Propionibacterium bacteria in commercial
scale [41, 42]. This method involves an anaerobic process in
which the highest amount of biomass is produced, then in the
second stage, aeration starts gently and Benzimidazole is
added. To increase the efficiency, the cells are transferred to
a new medium and better results are got compared to the
batch methods [43]. (Hunik, 2000). The bioproduction path
of this vitamin is well known in P. denitrificans, S.
typhimurium and P. Shermanii [44]. There are two ways for
vitamin B12 production in nature: the first is aerobic or
oxygen dependent in P. denitrificans and the second one is
anaerobic or oxygen independent in S. typhimurium, P.
shermanii and B. megaterium [45].
Vitamin B12 biosynthesis
2.2. The effect of stimulus factors
The effect of betaine on pseudomonas has been studied. 5
mg of betaine was added to the culture of bacteria and it was
abserved that vitamin B12 production increased by 20 times
[46]. In another study the role of amino acids, betaine and
choline in the bioproduction of vitamin B12 by
Propionibacterium species were investigated [47]. The whey
culture was enriched by 11 amino acids and then betaine
hydrochloride and choline chloride were added in different
porportions of 0.25, 0.5 and 0.75 % w/v. Among 11 amino
acids, Glutamic acid increased the growth of bacteria.
Betaine showed better stimulant effect compared to choline.
The stimulatory effect of Propionibacterium species on
vitamin B12 formation is similar to P. denitrificans [48, 49].
2.3. The effect of different carbon sources
Vitamin B12 is produced by using different carbon sources
such as molasses, sugar, sucrose and non-sugar sources [50,
51]. Coral investigated the effect of some carbon sources;
sugarcane molasses, lactate and glycerol) on propionic acid
fermentation. The maximum productivity and final
concentration of propionic acid was observed from lactate.
The propionic acid production from lactate was faster than
molasses due to not needing to be metabolized in the
glycolytic pathway. The other merit of using lactate to
glycerol and sugarcane molasses is that it does not require
pH control because of limited variation of pH. The highest
cell growth productivity, rate, concentration and yield of
produced biomass were observed after using sugarcane
molasses as substrate [52, 53]. A project was carried out to
determine the suitable concentration of lactose for
propionibacterium species growth in the lactose base culture.
The results showed that the suitable concentration of lactose
for the fermentation is nearly the same as its concentration in
skim milk and the fermentation did not increase by
increasing the lactose concentration [54]. Other results
revealed that propionic acid production in fermentation
culture increased by increasing the initial concentration of
lactose in batch fermentation. The initial concentration of
lactose should be more than 73 g/l in batch fermentation to
increase the concentration of propionic acid noticeably. It is
observed that P. freudenreichii subsp. Shermanii in culture
containing lactose showed lower efficiency in bacterial
growth stimulation and acid production compared to the
culture containing lactate [55]. Propionic acid production is
very variable from different sources and in different
temperatures. Temperature and the carbon source are both
effective in biomass production. The cellular function while
using cane molasses as a carbon source, were approximately
4 times more than using lactate however the propionic acid
production was similar. The final concentration of the
biomass in 30° C from cane molasses was more than other
sources. The optimum temperature for propionic acid was
30°C and for biomass production, it was 36°C. The best
carbon source for the bacteria to produce propionic acid and
biomass is lactate. However, using glycerol also leads in the
maximum propionic acid production [56].
The maximum propionic acid concentration was obtained by
using lactate at 30°C, because the conversion of lactate to
Current Nutrition and Food Sciences, 2018, Vol. 0, No. 0 3
propionic acid is easier and faster than converting cane
molasses to propionic acid [57]. To produce the equal
amount biomass, lower cane molasses consumed over the
lactate thus cane molasses is a more suitable source for the
propionic acid production. Therefore, it seems appropriate to
use cane molasses for the production of biomass in the first
phase and the consume lactate for propionic acid production.
On the other hand, propionic acid production by using cane
molasses in two steps, causes higher propionic acid
production along with increasing biomass production that are
useful in vitamin B12 production of [58]. Propionic acid
production decrease at 30 - 37°C but the biomass production
would increase. Propionibacterium is very pH sensitive and
grow slightly below pH=5 [59]. While the pH cannot be
controlled in a specific range, using lactate as a carbon
source is better than glycerol and cane molasses [60]. pH
changes more quickly through using glycerol and cane
molasses over the lactate. Acetic acid may reduce the quality
of the final product during the process. Therefore, glycerol as
a carbon source has this advantage. According to Fitz
equation, each mole of glycerol produced one mole of
propionic acid and one mole of acetic acid and water [61].
The efficiency propionic acid production is 0.64 moles per
glycerol mole in batch fermentation. In this conditions, lag
phase would be longer. Entering to logarithmic growth
phase, the consumption of glycerol and the propionic acid
production will increase. The cell growth stops after that the
propionic acid reaches to 15 g/l. This may be due to
decreasing of nutrients and also increasing the inhibitory
effect of propionic acid [62, 63].
2.4. Inoculum type and size
The bioproduction of vitamin B12 carried out by P.
denitrificans aerobically and by P. shermanii, P.
freudenreichii and S. typhimurium in anaerobica pathway.
Among all propionibacterium species are widely used for
microbial production of propionic acid. The highest
concentration of propionic acid was obtained in fermentation
by P. acidipropionici and the highest efficiency was
observed by P. freudenreichii [64, 65] and also the highest
amount of vitamin B12 was reported during fermentation of
molasses by P. freudenreichii [66]. P. freudenreichii is in the
GRAS list and also has the probiotics health properties.
Using P. freudenreichii and Lactobacillus acidophilus
simultaneously increase the propionic acid production [67,
68]. The volume of microorganisms added to the
fermentation culture is another important factor in the
process. This factor is often expressed as a volume percent.
Reducing the amount of inoculum under a certain level,
caused in prolonging adaptation phase and thus decreasing
the productivity. Increasing the amount of microorganisms
leads in sugar consumption and therefore cells accumulating
that reduces the final production. In various studies of
vitamin B12 fermentation, the inoculum rate was about 1 to
5% of the fermentation volume [69].
2.5. Fermentation time
The productivity of vitamin B12 reduced over time due to
the accumulation of inhibiting factors in culture. Many
studies have been done on the duration of process in vitamin
4 Current Nutrition and Food Science, 2018, Vol. 0, No. 0
B12 production. Various times (96, 120, 168 h) have been
applied in fermentation of this vitamin. The best results were
achieved in 96 hours [69, 70].
2.6. The effect of nitrogen source
Optimizing the environmental conditions in vitamin
production seems effective for increasing the production.
The type and concentration of nitrogen source also affect the
vitamin B12 production [71]. Nitrogen-containing
compounds may directly affect the secondary metabolisms.
Nitrogen is about 8-14% of the cells dry weight and it is
needed as a growth factor. Among the nitrogen sources for
fermentation, the yeast extract has been widely used in
production of organic acids and vitamin B12. The yeast
extract is obtained from the bakery’s yeast plasmolysis or
autolysis [55]. Corn syrup, the byproduct of starch extraction
from maize, is another source of nitrogen for fermentation
which is inexpensive and also contains a variety of essential
amino acids, vitamins and minerals. The nitrogen source
lower from a certain level would prolong the adaptation step
and the higher amounts may have the inhibitory effects on
growth [72, 73].
3. Fermentation system
Vitamin B12 have been produced by various methods such as
the continuous [74], batch system and fed-batch system [75]
in industry. By increasing the propionic acid production and
pH reduction, the efficiency would decrease in batch method
through time. Different methods are used to prevent the
inhibitory effect of propionic acid on cell growth using and
one of the most common ones is continuous fermentation
[65, 76]. Acid is removed by washing continuously in
continuous method, but the disadvantage of this method is
the excessive dilution being which can be solved by using
immobilized cells or filtration unit in reactor to recover cells
[77]. The usage of immobilized cells is more economical,
easier and also increases the efficiency over both batch and
fed batch methods. The fed batch fermentation is the most
common way for Vitamin B12 production. In fed batch
fermentation, mostly glucose would be added to the system
as the carbon source. Also the speed of adding the source
and as a result the availability of food for microorganisms
seems very important in fermentation process [78, 79].
3.1. Fed-batch Fermentation
Vitamin B12 has been produced by various methods,
including batch system, fed-batch system, and continuous
system on industrial scale [20, 80]. In the batch process, by
increasing propionic acid production and reducing pH, the
productivity decreased over time. In order to prevent the
inhibitory effect of propionic acid on the cell growth, several
methods have been used, which the continuous fermentation
is the common one [59]. (Ahmadi et al., 2016). In the
continuous method, the productivity would increase by
removing the acid through washing continuously, but the
problem is the excessive dilution, which can be solved by
recovering the cells inside the reactor, using immobilized
cells and also applying a filtration unit in the cell reactor
[81].
Khosravi et al.
Actually, the usage of immobilized cells is more convenient
and economical. Applying the immobilized cells increases
the productivity in both batch and fed-batch methods.
Totally the fed-batch is more preferable for vitamin B12
production than other methods [82].
CONCLUSION
Fermented dairy products contain valuable compounds,
which produced by the activity of various bacteria (vitamins,
propionic acid, conjugated linoleic acid, bioactive peptides).
Vitamin B12 is one of the most needed vitamins in the world
due to its valuable ability in some diseases treatment so this
essential nutrient biosynthesis seems very necessary. B12 is
produced commercially by the help of bacterial species and
propionibacterium plays an important role in the industrial
production. This bacterium is used widely for B12
biosynthesis and propionic acid production. Also more
progressive attempts are required for vitamin B12 production
by using modern biotechnological techniques due to lots of
disorders caused by vitamin deficiency. Propionibacteria are
so popular due to having interesting characteristics and being
economical. Among all bacteria the highest efficiency was
observed by P. freudenreichii. Optimizing the environmental
conditions in vitamin production seems effective for
increasing this vitamin production. The best carbon source
for this bacterium to produce biomass is lactate. Among the
nitrogen sources for fermentation, the yeast extract has been
efficient in production of organic acids and vitamin B12. The
suitable pH for growth of the Propionibacterium species is in
the range of 6 to 7 and the biomass production would
increase at 30 - 37°C. In the continuous method, the
productivity would increase by removing the acid through
washing continuously. Totally the fed-batch is more
preferable for vitamin B12 production than other methods. It
is recommended this research to be continued by evaluating
of the satiety properties of the processed product, evaluating
of the sensory properties of fermented dairy drinks produced
and also measuring of possibility of folate production in this
product by Propionibacterium.
CONFLICT OF INTEREST
The authors declare that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
The manuscript was written through contributions of all
authors.
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