LWT - Food Science and Technology 118 (2020) 108735

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LWT - Food Science and Technology

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Nutrient requirements of Lactobacillus casei Shirota and their application in T

fermented milk
Yanjiao Zhang, Li Meng, Mingzhi Ai, Yali Qiao, Gefei Liu, Xuejing Fan, Xuepeng Lv, Zhen Feng∗
Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, 600 Changjiang Road, 150030, Harbin, Heilongjiang,
China

A R T I C LE I N FO A B S T R A C T

Keywords: To shorten the fermentation time of fermented milk prepared with Lactobacillus casei Shirota, its nutrient re-
Lactobacillus casei Shirota quirements were analyzed. Based on nutrition requirements, fortified and unfortified milks were fermented by L.
Nutrient requirements casei Shirota and the fermentation time and qualitative characteristics of the final products were evaluated.
Fermented milk According to the nutrient requirement profile, milk was supplemented with Gly, Cys, Glu, Asn, Pro, Trp, pyr-
Fermentation time
idoxine, Ca-pantothenate, guanine, xanthine, Mg2+ and Mn2+. The time fermentation of milk fermented with L.
Quality
casei Shirota was shortened by 15 h when nutrient supplementation was added. Furthermore, the viable cell
count was improved in the presence of certain nutrients. No differences were observed in the flavor, as well as in
the textural and sensory properties, between fermented milk prepared with L. casei Shirota in the fortified and
unfortified milk. Our results indicated that specific nutrients promoted the growth of L. casei Shirota and reduced
the fermentation time of fermented dairy products prepared with L. casei Shirota.

1. Introduction
Lactobacillus casei, a commercial probiotic strain, can increase the
number of beneficial intestinal bacteria by balancing intestinal pro-
biotic bacteria and harmful bacteria. It can also lower cholesterol levels
(Aryana & Mcgrew, 2007; Wang et al., 2010). Yakult containing L. casei
Shirota shows antitumor, antibacterial and immunostimulatory activ-
ities (Sgouras et al., 2004). L. casei Shirota can improve chronic con-
stipation, enhance NK cell activity and modify allergen-induced im-
mune responses in allergic rhinitis (Dong, Rowland, Tuohy, Thomas, &
Yaqoob, 2010; Nagao, Nakayama, Muto, & Okumura, 2000). Further-
more, L. casei Shirota decreases Streptococcus mutans biofilm formation
on tooth surfaces by reducing biofilm acidogenicity and enamel lesion
depth to prevent cavity formation (Yadav et al., 2014). L. casei is used in
starter cultures and adjunct cultures for the intensification and accel-
eration of flavor development (Reale, Ianniello, Ciocia, Renzo, &
Mcsweeney, 2016) in fermented dairy products and for their beneficial
probiotic properties (Reale et al., 2015).
Presently, there are many fermented dairy products prepared with a
single probiotic strain on the Chinese market such as yakult (L. casei
Shirota), LAJOIE (L. paracasei LPC-37) and Cherita (L. rhamnosus GG
ATCC 53103). Most probiotics grow slowly in milk, and their acid-
ification rates are usually slow. Therefore, they cannot support fer-
mentation well in fermented milk (Shah, 2000). These fermented dairy
products prepared with a single probiotic strain require a long fer-
mentation time. The fermentation time of fermented milk prepared
with L. casei alone is long, and a long fermentation time is generally
unfavorable to the development of fermented milk prepared with a
single probiotic strain (Jeon, Seok, & Kwak, 2005). Furthermore, a long
fermentation time limits the use of L. casei in fermented dairy products
and other fermented foods. Therefore, shortening the fermentation time
of fermented milk prepared with L. casei should be considered.
The growth of L. casei requires a variety of nutrients that include
amino acids, vitamins, metal ions, buffers and other compounds (Aller
et al., 2014). But milk is lack of certain nutrients, their amounts are too
low to support the quickly growth of L. casei (Jeon et al., 2005). Ser and
Val promote L. casei respiration and biomass (Ricciardi, Ianniello,
Parente, & Zotta, 2015). Tryptone, yeast extract and glucose improve
the growth of L. casei (Oh, Rheem, Sim, Kim, & Baek, 1995). Sucrose is
the best carbon source for the production of lactic acid and the growth
of L. casei (Behbahani, Davoody, Shirani, & Mohabatkar, 2016). The
addition of Mn2+ can increase the tolerance of L. casei to oxidative
stress, thereby increasing the fermentation rate of L. casei (Ricciardi,
Ianniello, Parente, & Zotta, 2018). Unfortunately, systematic and global
studies of the nutrient requirements of L. casei and their application to
fermented milk have never been reported.
The purpose of this study was to shorten the fermentation time of
fermented milk prepared with L. casei Shirota by adding certain

∗
Corresponding author.
E-mail address: neau_fengzhen@163.com (Z. Feng).

https://doi.org/10.1016/j.lwt.2019.108735
Received 8 May 2019; Received in revised form 6 October 2019; Accepted 15 October 2019
Available online 18 October 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
Y. Zhang, et al.
nutrients to milk based on the nutrient requirements of L. casei Shirota.
In addition, the effects of the supplementation on the quality and sen-
sory properties of fermented milk prepared with L. casei Shirota were
evaluated.
2. Materials and methods
2.1. Strain, culture conditions and fermentation experiments
L. casei Shirota from Yakult poly-acid milk beverage was isolated,
purified and subcultured in a 250-mL Erlenmeyer flask containing
50 mL of MRS broth for 12 h at 37 °C (Savijoki, Suokko, Palva, &
Varmanen, 2010). The pellet was washed twice with PBS buffer
(50 mmol/L, pH 6.5), and the culture was fermented in a 10-L Biotech-
7000 bioreactor (Shanghai Baoxing, Shanghai, China) containing 7 L of
chemically-defined medium (CDM) with 1% (v/v) inoculum. The CDM
was prepared according to the method of Savijoki et al. (2010). The pH
was initially set at 6.50 in the uncontrolled pH cultures. The tempera-
ture and rotation speed were set at 37 °C and 150 rpm, respectively. The
growth rate was evaluated by spectrophotometric measurement at
650 nm. The biomass dry weight was determined gravimetrically. As
shown in Fig. 1a, The cells were harvested at 0, 3, 7, 10 and 12 h by
centrifugation (10,000×g, 10 min, 4 °C). Pellet and supernatant were
stored at −80 °C for further analysis. Batch fermentations were re-
peated three independent times.
2.2. Analysis of amino acids, vitamins, purines, pyrimidine and ions
Amino acid concentrations were determined by an amino acid
analyzer (Acquity UPLC; Waters Corp., Milford, MA, USA). The con-
centrations of thiamine, pyridoxine, Ca-pantothenate, folic acid, cya-
nocobalamin, biotin and nicotinamide were determined by RP-HPLC
with ultraviolet detector (Heudi & Fontannaz, 2005). The concentra-
tions of adenine, guanine, xanthine and uracil were measured by liquid
chromatography-tandem mass spectrometry (LC-MS/MS) according to
the method of Stentoft et al. (2014). The levels of K+, Na+, Mg2+, Fe2+
and Mn2+ were measured using an ion exchange method (Pohl &
Prusisz, 2007).
2.3. Effects of the nutrients on the growth of L. casei Shirota
The essential, nonessential and stimulatory nutrients were de-
termined by single omission experiments. A nutrient was considered
essential if its omission led to growth rates of less than half the max-
imum growth rate of the positive control, stimulatory when in its ab-
sence the growth rate was between 50% and 80% of that observed in
complete CDM, and nonessential when the growth rate in its absence
was 80% (or more) of that obtained in the complete CDM (Vera, Hebert,
Sesma, & Nader-Macías, 2009).
2.4. Selection of the nutrients
The nutrient consumption profile, necessary amino acid profile,
essential growth nutrients of L. casei Shirota, and the concentrations of
nutrients in unfortified milk were analyzed and compared. The nu-
trients with the high consumption rate, amino acids with the high ne-
cessary rate and essential growth nutrients were added to milk to
conduct the preliminary single addition experiments (Each of these
chosen nutrients was added to milk respectively for manufacturing the
fermented milk). The aim of the preliminary single addition experi-
ments was to select the nutrients which reduced the fermentation time
of fermented milk prepared by L. casei Shirota.
2.5. Preparation of fermented milk
L. casei Shirota was inoculated and propagated three times in 12%
2
LWT - Food Science and Technology 118 (2020) 108735
(w/v) sterile reconstituted skim milk at 37 °C. Subcultured L. casei
Shirota was inoculated at 2.0% (v/v) into milk and incubated at 37 °C
until the pH of the milk reached 4.5. The first sample was unfortified
milk (100 kg). According to the nutrients selected, fermented milks
were prepared with the milk which was added with Gly, Cys, Glu, Asn,
Pro, Trp, pyridoxine, Ca-pantothenate, guanine, xanthine, Mg2+ and
Mn2+. The second milk sample was fortified milk (100 kg). A total of
200 kg of fermented milk was manufactured from each fermention
batch. Batch fermentations were repeated three independent times.
2.6. Physicochemical, microbiological, sensory and textural analyses of
fermented milk
The water-retaining capability and titratable acidity were measured
as described by Jia, Chen, Chen, and Ding (2016). Textural properties
were analyzed by compression tests with the Texture Analyzer TA-Plus
(Lloyd Instruments, UK) texturometer as described by Miocinovic,
Miloradovic, Josipovic, Nedeljkovic, and Radovanovic (2016). The
flavor compounds were measured by SPME GC/MS as described by
Zareba, Ziarno, Scibisz, and Gawron (2014). The sensory properties of
the fermented milk were evaluated as described by Ahtesh,
Stojanovska, and Apostolopoulos (2017). A standard plate count pro-
tocol was applied to count the viable L. casei Shirota cells in which
fermented milk samples were serially diluted and plated on MRS agar,
followed by incubation at 37 °C for 48 h. The pH value of each sample
was measured at room temperature with a glass electrode attached to a
Jenway 3510 pH meter (Keison Products, Chelmsford, UK).
3. Results
3.1. Nutrient consumption profiles
Fig. 1a–c shows changes in the amino acid concentrations during
fermentation. The two highest consumed amino acids were Ser
(2.98 mmol/L) and Thr (2.48 mmol/L), followed by Val (1.52 mmol/L)
and Glu + Gln (1.48 mmol/L). The consumptions of Gly, Ala, Cys, Ile,
Leu, Tyr, Phe, Arg, Pro, Lys and Asp + Asn ranged from 0.5 to
1.39 mmol/L. The two least consumed amino acids were Met
(0.42 mmol/L) and His (0.46 mmol/L). Thr, Leu and Ile were consumed
at rates of 42.02%, 40.04% and 39.14%, respectively, followed by Phe
(30.03%) and Lys (28.78%). The consumption rates of Gly, Cys, Val,
Met, Tyr, Pro, Asp + Asn, Ser and Arg ranged from 23% to 28%. The
three lowest consumed amino acids were Ala (19.34%), His (21.24%)
and Glu + Gln (21%).
Fig. 1d shows changes in vitamin concentrations with increasing
culture time. The two highest consumed vitamins were myo-inositol
(0.0068 mmol/L) and nicotinic acid (0.0059 mmol/L), followed by p-
aminobenzoate (0.0052 mmol/L) and riboflavin (0.0051 mmol/L). The
consumptions of pyridoxine, biotin, thiamin and Ca-pantothenate
ranged from 0.0025 to 0.0048 mmol/L. The two least consumed vita-
mins were folic acid (0.002 mmol/L) and cyanocobalamin
(0.0006 mmol/L). Riboflavin and Ca-pantothenate were consumed at
rates of (38.12%) and (27.07%), followed by myo-inositol (24.51%)
and thiamin (20.82%). The consumption rates of pyridoxine, D-biotin,
nicotinic acid and folic acid ranged from 20.33% to 17.68%. The two
lowest consumed vitamins were cyanocobalamin (16.83%) and p-ami-
nobenzoate (14.1%).
Fig. 1e shows changes in the concentrations of purines and pyr-
imidines with increasing culture time. The highest consumption was
uracil (0.0807 mmol/L), followed by adenine (0.0655 mmol/L). The
lowest consumed purine was xanthine (0.0636 mmol/L), followed by
guanine (0.0549 mmol/L). The highest and lowest consumption rates
were 96.77% for xanthine and 83.03% for guanine. The consumption
rates of uracil and adenine were 90.44% and 88.47%, respectively. The
results indicated that purines and pyrimidines showed high consump-
tion rates. Fig. 1f–g shows changes in the metal ion concentrations with
Y. Zhang, et al. LWT - Food Science and Technology 118 (2020) 108735

(caption on next page)

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Y. Zhang, et al. LWT - Food Science and Technology 118 (2020) 108735

Fig. 1. Residual concentrations of amino acids (a, b, c), vitamins (d), purines and pyrimidines (e), minerals (f, g) and necessary amino acids (h) at different culture
time(t0 = 0 h, t1 = 3 h, t2 = 7 h, t3 = 10 h, t4 = 12 h). (a) Ser ( ), growth curve ( ); (b) Cys ( ), Glu + Gln ( ), Thr ( ), Val ( ), Ala ( ), Pro ( ), Lys
( ), Gly ( ), Asp + Asn ( ); (c) Met ( ), Ile ( ), Leu ( ), Tyr ( ), Phe ( ), His ( ), Arg ( ); (d) biotin ( ), thiamin ( ), riboflavin ( ), folic
acid ( ), pyridoxine ( ), myo-inositol ( ), nicotinic acid ( ), cyanocobalamin ( ), p-aminobenzoate ( ), Ca-panthothenate ( ); (e) adenine ( ), guanine
( ), uracil ( ), xanthine ( ); (f) Mg2+ ( ), Fe2+ ( ), Mn2+ ( ); (g) K+ ( ), Na+ ( ); (h) 0 h ( ), 3 h ( ), 7 h ( ), 10 h ( ) and 12 h ( ). The
results are means ± standard deviations of three independent experiments.

increasing culture time. The highest consumed metal ion was Na+ Table 1
(31 mmol/L), but its consumption rate was the lowest (13.59%), fol- Amounts of nutrients added to the milk.
lowed by K+ (3.25 mmol/L) and Mg2+ (0.164 mmol/L) at consumption Nutrient Milk Consumption Requirement Addition
rates of 26.25% and 23.09%, respectively. The consumption of Mn2+
was 0.1728 mmol/L, but its consumption rate was the highest (g/L) (g/L) (g/L) (g/L)
(97.35%). The lowest consumed metal ion was Fe2+ (0.0123 mmol/L), Arg 0.000551 0.1566 0.2484 0.2484
Leu 0.000961 0.2124 0.4549 0.4549
at a consumption ratio of 34.11%. Ile 0.000164 0.1881 0.2745 0.2745
Phe 0.000148 0.1833 0.6913 0.6913
Gly 0.000419 0.075 0.262 0.262
3.2. Necessary amino acid profiles Pro 0 0.1302 0.3023 0.3023
Val 0.003751 0.2075 1.084 1.084
Tyr 0.001305 0.1294 0.2411 0.2411
Based on amino acid concentrations in the whole-cell hydrolysates, Ser 0.000257 0.3636 0.1924 0.3636
the necessary amounts of the amino acids were investigated with re- Cys 0 0.194 0.648 0.648
spect to biomass at different culture time (Fig. 1h). The necessary Trp 0.00015 — — 0.56
amount of Ala accounted for 12.83% of the total amino acid require- Glu 0.05126 0.2675 0.6485 0.6485
Asn 0 0.217 0.4653 0.4653
ment, higher than that of other amino acids required for the growth of Riboflavin 0.00175 0.100679 — 0.100679
L. casei Shirota. The necessary amounts of Val, Glu + Gln and Ca-pantothenate 0.00346 0.104307 — 0.104307
Asp + Asn ranked second at 12.74%, 10.17% and 9.76%,respectively, Inositol 0.11 0.01862 — 0.01862
followed by Gly, Lys, Phe, Pro and Leu which ranged from 5% to 7% of Thiamine 0.00064 0.001041 — 0.001041
Pyridoxine 0.003152 — 0.003152
all amino acids required for the growth of L. casei Shirota.
Xanthine — 0.009677 — 0.009677
Guanine — 0.008303 — 0.008303
Uracil — 0.009043 — 0.009043
3.3. Effects of nutrients on the growth of L. casei Shirota Adenine 0.002728 0.008846 — 0.008846
Mg2+ 0.11 0.003985 — 0.003985
Mn2+ — 0.009493 — 0.009493
According to the single omission experiments, the nutrients were Fe2+ — 0.000687 — 0.000687
divided into three categories for the growth of L. casei Shirota: essential K+ 1.48 0.06175 — 0.06175
nutrients, stimulatory nutrients and non-essential nutrients. The results
are shown in Fig. 2. Arg, Phe, Leu, Ile, Val, Tyr, Ser, Cys, Trp, Ca-
pantothenate, pyridoxine, riboflavin, Mg2+ and glucose were essential 3.4. Selection of the nutrients
nutrients for the growth of L. casei Shirota. Pro, Gln, Met, Gly, nicotinic
acid, sodium acetate and Mn2+ showed growth-stimulating effects on A comparison of the aforementioned results with the nutrient con-
the growth of L. casei Shirota, whereas the absence of the other nu- centrations in milk showed that some nutrients were lacking in milk
trients had no obvious effects on the growth of L. casei Shirota. (Table 1). According to the consumption patterns of the nutrients and

Fig. 2. Effect of nutrient omission on the growth rate of L. casei Shirota. The columns represent the growth rates of L. casei Shirota when a nutrient was omitted from
the CDM compared with that in complete CDM. The growth rate was expressed as the percentage of the growth rate in complete CDM. The results are means ±
standard deviations of three independent experiments.

4
Y. Zhang, et al.
Fig. 3. Effects of nutrients that promote fermentation by L. casei Shirota (a, b, c), and the time it takes for L. casei Shirota to ferment nutrient-added milk (d). (a)
Control ( ), Cys ( ), Trp ( ), Ca-pantothenate ( ), formulation ( ); (b) control (
control ( ), xanthine ( ), Pro ( ), Mn2+ ( ) and Mg2+ ( ).
their concentrations in milk, the amino acids and vitamins with the
high consumption ratios (more than 25%) should be added to milk,
while the consumption ratios of other nutrients were over 20%.
Therefore, Arg, Leu, Ile, Phe, Lys, Thr, Tyr, Met, Val, riboflavin, Ca-
pantothenate, myo-inositol, thiamine, Mn2+, Fe2+, Mg2+, K+, xan-
thine, guanine, uracil and adenine were added to milk to conduct the
preliminary single addition experiments. According to the necessary
amino acid profiles and their concentrations in milk, the amino acids
with the high necessary ratios (close to or more than 8%) should be
added to milk. Therefore, Ala, Asn and Glu were added to milk to
conduct the preliminary single addition experiments. In addition, ac-
cording to the essential nutrients and growth-stimulating nutrients for
L. casei Shirota and their concentrations in milk, Gly, Ser, Cys, Trp, Pro
and pyridoxine were added to milk to conduct the preliminary single
addition experiments. The effects of the abovementioned nutrients on
the fermentation time of fermented milk prepared with L. casei Shirota
were evaluated by single addition experiments. Fig. 3a–c shows the
nutrients that promoted fermentation. Compared with control samples,
the fermentation time was shortened by at least 2 h when the selected
nutrients were added to milk. According to these results, Asn, Cys, Pro,
Glu, Gly, Trp, Ca-pantothenate, Mn2+, Mg2+, pyridoxine, guanine and
xanthine were simultaneously added to milk, and their concentrations
are shown in Table 1.
3.5. Effects of nutrients on the properties of fermented milk
The effects of the selected nutrients on the fermentation time, viable
5
LWT - Food Science and Technology 118 (2020) 108735
), Asn ( ), pyridoxine ( ), guanine ( ), Gly ( ), Glu ( ); (c)
cell count, water-retaining capability, titratable acidity, textural prop-
erties, flavor compounds and sensory properties of fermented milk
prepared with L. casei Shirota were evaluated. The results are shown in
Table 2, Fig. 4a and Fig. 4b. Compared with unfortified milk, the fer-
mentation time of fermented milk prepared with L. casei Shirota sup-
plemented with the nutrients was shortened by approximately 15 h. The
viable cell count of fermented milk prepared with L. casei Shirota
supplemented with nutrients was twice that of the unfortified milk. No
differences in typical flavor compounds, acceptability, taste, hardness,
adhesiveness, springiness and cohesiveness were observed between the
unfortified milk and fortified milk. In addition, changes in the viable
cell count and pH of fermented milk were determined during storage at
4 °C (Fig. 4c). Compared with unfortified milk, the percentage of viable
L. casei Shirota cells was high in fortified milk at the end of the 28-d
storage period. A pH decline of 0.5–0.6 units was observed in the fer-
mented milk at the end of the 28-d storage period at 4 °C.
4. Discussion
A shortage of nutrients is the main limiting factor for the growth of
lactic acid bacteria (LAB) (Youssef, Goma, & Olmos-Dichara, 2005).
Solving the nutrient-limiting conditions is a very effective way to im-
prove the fermentation process (Zalan, Hudacek, Stetina, Chumchalova,
& Halasz, 2010). The addition of these nutrients to milk promoted the
growth of L. casei. Although the growth of heterolactic bacteria has
been studied under various conditions, including variations in the pH
and in the concentrations of initial substrates and lactic acid, only a few
Y. Zhang, et al. LWT - Food Science and Technology 118 (2020) 108735

Cohesiveness (g)

754 ± 29.41
792 ± 26.12
Springiness (g)

965 ± 34.78
985 ± 29.36
Adhesiveness (g)

−71 ± 5.47
−84 ± 6.09
103.05 ± 0.79
106.53 ± 0.92
Hardness (g)
Water-retaining Capability (%)

32.45 ± 0.02
36.26 ± 0.02
Titratable Acidity (°T)

70 ± 6.25
79 ± 6.63
Viable counts ( × 109 cfu/mL)

1.27 ± 0.16
2.47 ± 0.36

Fig. 4. The typical volatile flavor compounds in fortified and unfortified fer-
mented milk (a). Fortified fermented milk ( ), unfortified fermented milk (
). Graphic representation of the mean sensory evaluation by quantitative de-
Fermentation time (h)

scriptive analysis of fortified and unfortified fermented milk (b). Control sample
( ), Fortified sample ( ). Changes in the cell viable counts of L. casei
Shirota and pH in fortified and unfortified fermented milk during storage at 4 °C
Properties of the fermented milk.

(c). Cell viable counts in fortified sample ( ), cell viable counts in control
31 ± 1.67
16 ± 1.5

sample ( ), pH of fortified sample ( ), pH of control sample ( ).

studies have provided detailed analyses of the nutritional medium

limitations from a kinetic perspective. The present study highlighted
Fortified sample

the necessity of certain nutrients for the growth of L. casei Shirota. If

Control sample

these nutrients were supplied insufficiently, the efficient growth of L.

casei Shirota was not achieved. Furthermore, nutrients with high con-
Sample
Table 2

sumption rates, as well as necessary and essential nutrients, should be

considered in fermented foods prepared with L. casei Shirota to shorten

6
Y. Zhang, et al.
the fermentation time. In this study, the fermentation time of fermented
milk was shortened by 15 h by adding certain nutrients.
The addition of Cys, Asn, Trp, Pro, Gly, Glu, guanine and xanthine
to milk shortened the fermentation time of the fermented milk prepared
with L. casei Shirota. Based on the L. casei genome, the metabolic
profiles of Asn, Cys, Pro, Trp, Gly, Glu, guanine and xanthine have been
reported (Elena et al., 2014). The results indicated that L. casei has the
ability to synthesize Pro, Cys, Gly, guanine and xanthine. Cys and Gly
are synthesized by Ser, whereas Pro is produced by the decomposition
of Glu. However, these synthesis pathways are very complicated. In
addition, L. casei cannot synthesize Glu, Asn, Ser, Trp, guanine and
xanthine through a de novo pathway. The substrates for the synthesis of
these nutrients were deficient in milk, or the synthesis rates of these
nutrients did not meet the growth requirements of L. casei. However,
these nutrients were important to the growth of L. casei and reduced the
fermentation time of the fermented milk prepared with L. casei. In ad-
dition, Asn, Pro, Gly and guanine were mostly consumed from the mid-
exponential growth phase to the end-exponential growth phase. Ac-
cordingly, these nutrients promoted fermentation mainly from the mid-
exponential growth phase to the end-exponential growth phase in fer-
mented milk. Asn and Pro improved the growth of L. casei (Ricciardi
et al., 2015). These results show that Pro stimulated the growth of L.
casei. Furthermore, Pro is a key nitrogen source for cell growth and
metabolism (Farvin, Baron, Nielsen, Otte, & Jacobsen, 2010). Gly can
affect Lactobacillus growth (Grobben et al., 1998). Most Lactobacilli are
auxotrophic for purines (Kilstrup, Hammer, Jensen, & Martinussen,
2010). Guanine can significantly stimulate the growth of Lactobacilli in
whole and skim milk (Elli, Zink, Reniero, & Morelli, 1999). In addition,
Mn2+ and Ca-pantothenate can promote the fermentation process
mainly from the mid-exponential growth phase to the end-exponential
growth phase in fermented milk. Mn2+ improves oxygen tolerance,
resistance to oxidative stress and biomass production in L. plantarum
(Ricciardi et al., 2018). Ca-pantothenate participates in coenzyme
biosynthesis, and it is essential for the growth of Lactobacilli (Hebert,
Raya, & Giori, 2000). Glu, Cys, Trp, pyridoxine, Mg2+ and xanthine
promoted fermentation mainly from the lag phase to the mid-ex-
ponential growth phase in fermented milk. They are likely to promote
the growth and metabolism of L. casei in the lag growth phase and in
the early exponential growth phase as stimulating factors. Furthermore,
Cys (a sulfur-containing amino acid) and Trp are required for Lacto-
bacillus growth, whereas Glu promots Lactobacillus growth (Grobben
et al., 1998). These results show that Trp is an essential nutrient for the
growth of L. casei. Mg2+ can affect the enzyme activity, nitrogen me-
tabolism and lag time of bacterial growth (Yang et al., 2017). Pyr-
idoxine is required for deamination, transamination and decarboxyla-
tion reactions (Teusink et al., 2005). The physiological functions and
requirements of these nutrients may be the main reasons for promoting
the fermentation of fermented milk.
Aroma and flavor are important properties for consumer accep-
tance. The bitterness score of fermented milk prepared with L. casei
Shirota in the presence of nutritional supplementation was only slightly
higher than that of unfortified milk. This might have been due to the
addition of amino acids (Ahtesh et al., 2017). The panelists did not
detect any significant difference in the aroma between the fortified milk
and the unfortified milk. The SPME GC/MS can be used to assist in the
sensory evaluation of the overall flavor characteristics of fermented
milk. The results from the SPME GC/MS study, which characterized
flavor compounds produced in the fermented milk, are similar with
those of a previous study (Zareba et al., 2014). The concentrations of
flavor compounds were somewhat different, which was attributed to
the different strains in the fermented milk. The results indicated that
the addition of nutrients had no adverse effect on the quality and flavor
of fermented milk. The initial viable L. casei Shirota cell count largely
exceeded the minimum therapeutic threshold of 106 cfu/mL (Varga,
Süle, & Nagy, 2014). The survival rate of L. casei Shirota in fermented
milk in the presence of certain nutrients was higher than that of
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LWT - Food Science and Technology 118 (2020) 108735
unfortified milk. In other words, the nutrients could improve L. casei
Shirota cell viability in fermented milk prepared with the necessary
nutrients. Glu can improve the acid resistance of LAB and protect LAB
from damage caused by a low pH environment (Guan et al., 2013).
Mg2+ can enhance the survival rate of Lactobacillus (Yang et al., 2017).
The survival rate of L. casei during 28 d of storage was similar to that of
L. acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12 in cow
milk (Varga et al., 2014).
5. Conclusion
This study provided a reliable foundation for further studies by in-
tegrating the nutrient limitations and requirements of L. casei Shirota
and the fermented milk using L. casei Shirota. Various nutrients,
namely, Asn, Cys, Pro, Gly, Glu, Try, guanine, Ca-pantothenate, pyr-
idoxine, Mn2+ and Mg2+ were added to milk for fermentation with L.
casei Shirota, and the fermentation time was shortened by 15 h. The
addition of these nutrients had no obvious effect on the textural, sen-
sory and flavor properties of fermented milk prepared with L. casei
Shirota. The viable cell count, water-retaining capability and titratable
acidity of the fermented milk prepared with L. casei Shirota in the
fortified milk were improved. We developed a new milk media for
fermented milk prepared solely with L. casei Shirota after adding certain
nutrients to milk, and the probiotic fermented milk achieved good
properties within a shorter fermentation time.
Declaration of competing interest
It should be understood that none of the authors have any financial
or scientific conflict of interest with regard to the research described in
this manuscript.
Acknowledgments
This work was supported by grants from the National Key Research
and Development Plan Project (2018YFD0400405), National Natural
Science Foundation of China (31771989) and Natural Science
Foundation of Heilongjiang Province (C2016023).
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