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Lactic acid bacteria-containing chocolate as a practical probiotic product with increased acid tolerance
Author’s Accepted Manuscript Lactic acid bacteria-containing chocolate as a practical probiotic Product with increased acid tolerance Yasunori Yonejima, Keiko Hisa, Marina Kawaguchi, Hiroaki Ashitani, Toshiyuki Koyama, Yoko Usamikrank, Nayumi Kishida, Shigenobu Kishino, Jun Ogawa
PII: DOI: Reference:
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S1878-8181(15)00119-X http://dx.doi.org/10.1016/j.bcab.2015.09.001 BCAB298
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 14 July 2015 Revised date: 4 September 2015 Accepted date: 8 September 2015 Cite this article as: Yasunori Yonejima, Keiko Hisa, Marina Kawaguchi, Hiroaki Ashitani, Toshiyuki Koyama, Yoko Usamikrank, Nayumi Kishida, Shigenobu Kishino and Jun Ogawa, Lactic acid bacteria-containing chocolate as a practical probiotic Product with increased acid tolerance, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2015.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Lactic Acid Bacteria-Containing Chocolate as a Practical Probiotic Product with Increased Acid Tolerance
Author names and affiliations Yasunori Yonejima a , Keiko Hisa a , Marina Kawaguchi a , Hiroaki Ashitani b , Toshiyuki Koyama b , Yoko Usamikrank b , Nayumi Kishida c , Shigenobu Kishino c and Jun Ogawa c *
a
Research and Development Department, Nitto Pharmaceutical
Industries, Ltd., 35-3 Minamibiraki, Kamiueno-cho, Muko 617-0006, Japan b
Central Laboratory, LOTTE Co., Ltd., 3-1-1 Numakage Minami-ku,
Saitama 336-8601, Japan c
Division of Applied Life Science, Graduate School of Agriculture ,
Kyoto University, Kyoto 606-8502, Japan
* To whom correspondence should be addressed . Fax: +81-75-753-6113; E-mail: [email protected]
Abstract This study shows an evaluation of the method to improve acid tolerance of probiotic bacteria aimed at enhancement of the probiotic effects in the intestinal canal. In the tolerance test of simulated digestive juice in vitro, the viable rate of Lactobacillus brevis subsp. coagulans (Labre) processed with
chocolate
(Labre-in-chocolate)
is
significantly
increased,
approximately one hundred times higher, compared to those of freeze -dried Labre powder and Labre contained in beverage. This pro tective effect 1
depends on the content of water in chocolate for the probiotics processing. Differences in tolerance of metabolic activity were investigated with Labre-in-chocolate and freeze-dried Labre powder, both treated with simulated
digestive
Labre-in-chocolate
juice
remained
in
vitro.
significantly
The
enzyme
even
though
activity that
of
of the
freeze-dried Labre powder was inactivated. These results indicate that chocolate processing is an effective method making probiotics to be delivered to the intestine in a viable condition and to be e ffective for host health.
Key words: Lactobacillus brevis subsp. coagulans; Lactobacillus brevis NTM003; tolerance test of simulated digestive juice ; chocolate
1. Introduction
A bacterium that provides specific health benefits to the human body is called a probiotic, which is expected to have various benefits for health maintenance when consumed as a food component or supplement. Lactobacillus brevis subsp. coagulans called Labre is a lactic acid bacterium that belongs to probiotics. Labre is effective to improve the immune system through inducement of interferon-α production and enhancement of natural killer (NK) cell activity (Kishi et al., 1996; U.S.P.5662900). In other cases, it has been reported that Labre has various benefits for the human body such as early int ervention in irritable bowel syndrome (Murakami et al., 2012), activation the availability of medical herb flavonoid s (Sakurama et al., 2014). Moreover, there is a report that Labre could reduce the risk of infection in children 2
during influenza season (Waki et al., 2014). As discussed above, the effectiveness of probiotics such as Labre is now widely accepted by consumers, and this may play a part in the growth element of the functional food market. Lactic acid bacteria, however, are damaged by gastrointestinal stresses in the case of oral intake, and the viable rate is dramatically decreased in the intestine. It is expected to deliver live bacteria into the intestine to maintain the activities of the probiotics themselves or to protect their metabolisms generating effective compounds. Therefore, a live-bacteria-containing pharmaceutical preparation is coated with an enteric coating agent to prevent the pharmaceutical from being exposed to gastric acid aimed at enhancement of the probiotic effect s in the intestine. Lactic acid bacteria are usually taken with yogurts and beverages, but they are easily exposed to gastric acid. In this case, the probiotic effect s may not be expressed adequately in the intestine. The aim of this study is to establish the practical methods to deliver bacteria to the intestine in a viable condition in the form of general food through the verification of increased acid tolerance of Labre, which is coated
with
chocolate
against
simulated
digestive
juice
in
vitro.
Furthermore, we confirmed sustained enzyme activity in the lactic acid bacteria-in-chocolate after the treatment with simulated digestive juice with nucleoside-hydrolyzing activity as an indicator.
2. Materials and methods
2.1. Lactic acid bacteria Lactobacillus brevis subsp. coagulans (Labre) FERM BP-4693 used in this study is isolated from suguki zuke (a regional pickle of Japan made from a 3
turnip-like vegetable) in Kyoto, Japan, and the probiotic powder was produced at Nitto Pharmaceutical Industries, Ltd. (Kyoto, Japan). Labre was incubated at 30℃ for 24-48 h in a growth medium mainly composed of 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L meat extract. The culture supernatant was removed by centrifugation . The resultant bacterial cells were freeze-dried and used as Labre powder. Lactobacillus brevis NTM003 (NTM003) NITE BP-1634 used in this study is isolated from nanohanazuke (a regional pickle of Japan made from the flower of cole) in Kyoto, Japan, and the probiotic powd er was produced at Nitto Pharmaceutical Industries, Ltd.. NTM003 was incubated at 37℃ for 24-48 h in a growth medium mainly composed of 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L meat extract. The culture supernatant was removed by centrifugation . The resultant bacterial cells were freeze-dried and used as NTM003 powder. Commercial probiotics used in this study are as follows: Product A is a probiotic beverage that contains Lactobacillus brevis. Product B is a probiotic beverage that contains Lactobacillus casei. Product C is a probiotic yogurt that contains some kind of lactic acid bacteria and is certified as a Food for Specified Health Uses in Japan. Product D is a world-wide consumed probiotic yogurt that contains some kinds of lactic acid bacteria.
2.2. Preparation of Labre-in-chocolate and NTM003-in-chocolate Labre or NTM003 powder was added to melted chocolate, and then this highly concentrated lactic acid bacteria-in-chocolate was taken into tempered chocolate, keeping the temperature around 32℃ . This chocolate liquid was poured into a mold, cooled, and used as probiotics-in-chocolate. Final concentration of Labre or NTM003 powder in the chocolate was 0.5% 4
(w/w). The used chocolate was a commercial chocolate (LOTTE Co., Ltd. Saitama, Japan) composed of 30% (w/w) cocoa components (cocoa mass and cocoa butter), 20% (w/w) milk component, 9% (w/w) vegetable fat component, 40% (w/w) sugar component, and 0.5% (w/w) emuls ifier and natural vanilla flavoring components.
2.3. Tolerance of lactic acid bacteria against simulated digestive juice in vitro A simulated digestive juice was made as described previously (Azuma et al., 2001) with modifications. Briefly, Lactobacilli MRS broth (Becton Dickinson, and Company, NJ, USA) with 0.04% (w/v) pepsin (Wako, Osaka, Japan)
was
adjusted
to
pH
2.5
using
HCl.
Four
gram
of
each
probiotics-in-chocolate were added to 300 ml of simulated digestive juice and shaken at 37℃ for a few hours at 70 rpm; the liquid surface was gently shaken by the revolutions per minute. Then the total viable count of probiotics was analyzed after 0.5 h, 1 h, and 2 h incubations in simulated digestive juice by sampling 1 ml of the solution, dil uting adequately, and plating on MRS broth plates with 1.5% (w/v) agar. For the treatment of probiotics powders or commercial probiotics products, the initial viable count
before
the
treatment
was
adjusted
as
many
as
it
of
probiotics-in-chocolate.
2.4. Probiotics-in-chocolate with fresh cream or freeze -dried cream Probiotics-in-chocolate with fresh cream was made as follows. Boiled fresh cream (20 g) was added to chopped chocolate (LOTTE Co., Ltd., Saitama, Japan) (50 g), and melted by heating at 40 ℃ . Then 0.5% (w/w) probiotics powder was added and mixed quickly. This chocolat e liquid was poured into a tray and cooled at 4℃ for 0.5 h. 5
Probiotics-in-chocolate with freeze-dried cream was made as follows. A chopped chocolate (50 g) was melted by heating at 40℃ . Then freeze-dried cream (10.4 g) and 0.5% (w/w) probiotics powder was added and mixed quickly. This chocolate liquid was poured into a tray and cooled at 4 ℃ for 1 h.
2.5. Activity analysis of the enzyme converting purine nucleosides into purine bases The enzyme activity of probiotics converting purine nucleosides into purine bases was assayed as described previously (Tsuboi et al., 2012; J.P.5149305) with modifications. Briefly, 50 mg of probiotics powder or 1 g of probiotics-in-chocolate which contained 50 mg of probiotics powder after treatment with 0.85% NaCl or simulated digestive juice was washed twice with 0.85% NaCl. After washing, 4.0 ml of the nucleoside solutions containing 2 mM guanosine and 4 mM inosine in 0.1 M potassium phosphate buffer (pH 7.0) were added into the resulting probiotics precipitation. The mixture was incubated for 1 h at 37°C, and then the reaction was terminated by adding 0.5 ml of 2.0 N HCl. Guanine and hypoxanthine
in
the
reaction
mixtures
were
quantified
with
high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a Shimadzu LC 10A -VP system (Shimadzu, Kyoto, Japan) equipped with a Cosmosil 5C18 -PAQ column (4.6 × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) at 40°C. 10 μl of the filtered supernatant of the reaction mixture was injected to HPLC. The mobile phase was 50 mM KH 2 PO 4 (pH 3.0). The flow rate was 1.0 ml/min. The effluents were monitored at 254 nm using diode array detector (SPD -M20A, Shimadzu Kyoto, Japan).
6
2.6. Statistics Each result is expressed as the mean ±standard deviation (SD). Tukey’s multiple comparison tests o r S t u d e n t ’ s t - t e s t w e r e u s e d t o c o m p a r e e a c h g r o u p . Statistical analyses were done with Excel Toukei 2010 (Social Survey Research Information, Tokyo, Japan).
3. Results
3.1 Tolerance of differently processed probiotics against simulated digestive juice The acid tolerances of various probiotics in different preparations, i.e., Labre powder, Labre-in-chocolate, and Labre-in-beverage (Product A), were evaluated.
Fig. 1 shows the tolerance of lactic acid bacteria against
simulated digestive juice in vitro. The viable count of Labre powder was not stable; it immediately decreased from 10 6 cfu/ml to 10 3 cfu/ml in 0.5 h, notably decreased to 10 cfu/ml in 1 h, and finally was non-detected (marker not shown) in 2 h after adding into simulated digestive juice.
The viable
count of Labre-in-beverage (Product A) was also not stable: it immediately decreased from 10 6 cfu/ml to 10 3 cfu/ml in 0.5 h, notably decreased to 10 cfu/ml in 1 h, and finally decreased to 10 cfu/ml in 2 h after adding to simulated digestive juice. However, the viable count of Labre-in-chocolate was more stable for at least 2 h, it decreased slightly to 10 5 cfu/ml in 2 h after adding to simulated digestive juice. These results showed that the viable rate of Labre-in-chocolate is significantly increased , by at least one hundred
times,
compared
to
those
of
the
Labre
powder
and
Labre-in-beverage.
3.2 Effects of chocolate processing on different Lactobacillus brevis strains 7
Viability-enhancing effects of chocolate processing were investigated with two representative Lactobacillus brevis strains.
Fig. 2 shows a
comparison of tolerance against simulated digestive juice in vitro between Lactobacillus brevis subsp. coagulans (Labre) and Lactobacillus brevis NTM003 (NTM003). The tolerance test of lactic acid bacteria against simulated digestive juice in vitro was conducted as stated above. The viable rate
of
the
Labre-in-chocolate
was
stable,
and
th at
of
the
NTM003-in-chocolate was also stable. However, the viable rate of Labre powder was lower than that of NTM003 powder after 1 h. These results showed that the viable rates in simulated digestive juice differ in each of lactic acid bacterial strains, but the tolerances against simulated digestive juice were improved by adding them to chocolate.
3.3 Comparison of tolerance between Labre -in-chocolate and several probiotic foods Fig. 3 shows a comparison of tolerance against simulated digestive juice in vitro between Labre-in-chocolate and several probiotic foods. The tolerance test of lactic acid bacteria against simulated digestive juice in vitro
was
conducted
as
stated
above.
The
viable
count
of
Labre-in-chocolate was stable for at least 2 h in simulated digestive juice. On the other hand, the viable count s of Product B and Product D decreased gradually, and the viable count of Product C de creased immediately and was not detected (marker not shown) after 0.5 h in simulated digestive juice.
3.4 Effects of fresh cream and freeze-dried cream on protective effects of chocolate Fig. 4 shows a comparison of tolerance against simulated digestive juice between Labre-in-chocolate added with fresh cream or freeze -dried cream. 8
The tolerance test of lactic acid b acteria against simulated digestive juice in vitro
was
conducted
as
stated
above.
The
viable
counts
of
Labre-in-chocolate and those of Labre-in-chocolate added with freeze-dried cream
were
stable
for
at
least
2
h.
But
the
viable
count
of
Labre-in-chocolate added with fresh cream showed different results: it immediately decreased from 10 6 cfu/ml to 10 4 cfu/ml in 0.5 h, and after that it was kept at the same level at least 2 h. These results showed that the viable rate of Labre significantly decreases, by about ten times, by addition of fresh cream but not freeze-dried cream. These results indicated that the water in fresh cream affected protecting effects of chocolate on probiotics.
3.5 Effects of chocolate processing on the stability of the enzyme in probiotics converting purine nucleosides to purine bases Table 1 shows a comparison of enzyme activity converting purine nucleosides to purine bases between Labre powder and Labre-in-chocolate after treatment with simulated digestive juice. Control experiments were carried out after treatment with 0.85% NaCl instead of simulated digestive juice. Reactions were carried out with 2 mM guanosine and 4 mM inosine as substrates. In the reaction with Labre powder after treated 2 h with 0.85% NaCl, generations of 0.208 mM guanine and 0.489 mM hypoxanthine derived from decomposition of the substrates were observed after the reaction, but no generations of guanine and hypoxanthine were observed in the reaction with Labre powder treated by simulated digestive juice 2 h. On the other hand, in the reaction with Labre-in-chocolate treated 2 h with 0.85% NaCl, generations of 0.446 mM guanine and 1.316 mM hypoxanthine were observed, and
generations of 0.320 mM guanine and 0.838 mM
hypoxanthine were observed in the reaction with Labre-in-chocolate treated 2
h
with
simulated
digestive
juice. 9
These
results
showed
that
Labre-in-chocolate retained the enzyme activity in 0.85% NaCl and also in simulated digestive juice, indicating that chocolate processing is useful to stabilize the enz yme activity in probiotics during gastric acid treatment.
4. Discussion A large number of health-promoting effects of probiotics have been reported, and it commonly works on multiple people, however, it is reasonable to postulate that the majority of probiotics do not colonize in the intestine (Sanders, 2011) but is capable of growing during passage through the gastrointestinal track. A large population of living probiotics may be beneficial to effect host health as transient bacteria. A variety of studies have been conducted to demonstrate the positive effects of probiotics. Currently, metabolites produced by probiotics attract considerable attention among biochemists. Short-chain fatty acids produced by probiotics can act as metabolic regulators of energ y uptake in adipose tissue (Kimura et al., 2013). Lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition (Kishino et al., 2013). It is essential to deliver bacteria in a viable form to expect the effects of metabolites and enzymatic activity. Probiotics are commonly available in foods such as yogurt, beverages, freeze-dried powders, and tablets, but probiotics in these foods are not protected against gastric acid. Regarding oral medicines in the for m of powder, tablets and capsules are protected against gastric acid by an antacid or enteric coating agent. If such protection methods are easily applied in the manufacturing process of general foods, people can consume probiotic s that are delivered to the intestine in a viable condition. In this study, we aimed to establish practical and efficient methods to enhance the tolerance of probiotics to gastric acid. We observed the 10
increased viable rate of probiotics by coating with chocolate against simulated digestive juice in vitro. The fasting gastric pH value is generally 1 to 2, and it increases to 4 to 5 after eating and drink ing. It takes about 2 h for digested material to pass from the stomach to the duodenum, hence we set the pH value as 2.5 and 2 h duration as the conditions of the test. In the tolerance test in vitro, the viable rate of Labre coated with chocolate (Labre-in-chocolate) is significantly increased , approximately one hundred times higher, compared to those of Labre powder and Labre-in-beverages. It is considered that chocolate coats the surface of Labre to hardly soluble digestive juice and protect s Labre from the stress of low pH. We compared the effect of acid tolerance between Labre and NTM003, which is primarily stronger than Labre. The result showed that Labre-in-chocolate has effectiveness equal to that of NTM003 against simulated digestive juice. This result indicates that Labre-in-chocolate exerts an increased stability against gastric acid regardless of inh erent strength. Moreover, Labre-in-chocolate shows better tolerance against simulated digestive juice than that of probiotic yogurts on the market. To investigate the effect of water content in chocolate on protection against simulated digestive juice, we compared the tolerance between Labre-in-chocolate added with fresh cream or freeze-dried cream, which contains 10% and just a few amount of water, respectively. The viable rate of Labre-in-chocolate added with fresh cream is lower than th at in normal Labre-in-chocolate and Labre-in-chocolate added with freeze-dried cream. This result indicated that protection against simulated digestive juice diminished by water, which decreased the water-repellent effects may be caused by cacao lipids in the chocolate. Furthermore, we compared the enzyme activity converting purine nucleosides
into
purine
bases 11
between
Labre
powder
and
Labre-in-chocolate after treatment with digestive juice. The enzyme activity contributes to inhibiting the absorption of purine bodies (Tsuboi et al., 2012; J.P.5149305) and reduces serum uric acid contents. The enzyme activity of Labre-in-chocolate remains considerable level to maintain its biochemical effects, even though the Labre powder was lost the enzymatic activity. These results indicated that the expression of enzyme activity of Labre depends on increased viability in intestine caused by chocolate processing. In conclusion, chocolate processing is an effective way to deliver probiotics in a viable form to the intestine. Lipid s in chocolate may protect probiotics, are degraded by lipase in the intestine, and then release probiotics into intestine as a viable form and make them effective to the host health. Further studies to clarify the mechanisms of the protection are required.
5. Conclusions It is essential to deliver live bacteria into the intestine to maintain the activities as of the probiotics. Acid stress in stomach is one of the main causes of deactivation of live bacteria. So, we evaluated the method to improve acid tolerance of probiotic bacteria by coating in with chocolate. Chocolate processing was found as an effective way to deliver probiotics in a viable form to the intestine. Probiotics-in-chocolate was found to be more stable against gastric acid treatment than probiotics powder and those in beverages or in yogurt.
Chocolate processing was also useful to keep the
enzymatic activities of the probiotics against gastric acid treatment.
We
hope that this method improve the qualities of the probiotics contributing to human health and welfare.
12
Acknowledgment This work was supported, in part, by Scientific Research Grants in Aid 23688011 (to S.K.), and 26660064, 23248014 (to J.O.).
Reference Azuma, Y., Ito, K., Sato, M., 2001. Simulated digestive juice tolerance and inhibitory effect on harmful intestinal bacteria of Lactobacillus gasseri NY0509 and Lactobacillus casei NY1301. Nippon Shokuhin Kagaku Kogaku Kaishi. 48 (9), 656-663. Kimura, I., Ozawa, K., Inoue, D., Imamura, T., Kimura, K., Maeda, T., Terasawa, K., Kashihara, D., Hirano, K., Tani, T., Takahashi, T., Miyauchi, S., Shioi, G., Inoue, H., Tsujimoto, G., 2013. The gut microbiota
suppresses
insulin -mediated
short-chain
fatty
receptor
acid
GPR43.
fat
accumulation
Nat.
Commun.
via 4,
the DOI:
10.1038/ncomms2852. Kishi, A., Uno, K., Matsubara, Y., Okuda, C., Kishida, T., 1996. Effect of the oral administration of Lactobacillus brevis subsp. coagulans on ineterferon-alpha producing capacity in humans. J. Am. Coll. Nutr. 15 (4), 408-412. Kishino, S., Takeuchi, M., Park, S.B., Hirata, A., Kitam ura, N., Kunisawa, J., Kiyono, H., Iwamoto, R., Isobe, Y., Arita, M., Arai, H., Ueda, K., Shima, J., Takahashi, S., Yokozeki, K., Shimizu, S., Ogawa, J., 2013. Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc. Natl. Acad. Sci. U. S. A. 110 (44), 17808-17813. Murakami, K., Habukawa, C., Nobuta, Y., Moriguchi, N., Takemura, T., 2012. The effect of Lactobacillus brevis KB290 against irritable bowel syndrome:
a
placebo-controlled 13
double-blind
crossover
trial.
Biopsychosoc. Med. 6 (16), DOI: 10.1186/1751 -0759-6-16. Sakurama, H., Kishino, S., Uchibori, Y., Yonejima, Y., Ashida, H., Kita, K., Takahashi, S., Ogawa, J., 2014. β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to gly coside hydrolase family 30. Appl. Microbiol. Biotechnol. 98 (9), 4021-4032. Sanders, M.E., 2011. Impact of probiotics on colonizing microbiota of the gut. J. Clin. Gastroenterol. 45 , S115-S119. Tsuboi, Y., Kaneko, N., Sato, A., Lactic acid bacterium havin g effect of lowering blood uric acid level, J . Pat. No. 5149305. Tsunataro K., 1997. Method of Increasing interferon production in humans. U.S. Pat. No. 5662900. Waki, N., Matsumoto, M., Fukui, Y., Suganuma, H., 2014. Effects of probiotic Lactobacillus brevis KB290 on incidence of influenza infection among schoolchildren: an open -label pilot study. Lett. Appl. Microbiol. 59, 565-571.
Figure legends Fig. 1. Comparison of tolerance against simulated digestive juice in vitro between Labre powder, Labre-in-chocolate, and Labre-in-beverage (Product A). Each value is the mean±SD (n=3). The value of Labre powder at after 2 h was not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b
Different letters represent a
significant difference between groups; p<0.01. ○, Labre powder; △, Labre-in-chocolate; □, Labre-in-beverage (Product A).
Fig. 2. Comparison of tolerance against simulated digestive juice in vitro 14
between L. brevis strains. Each value is the mean±SD (n=3). The value of Labre powder at after 2 h was not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b , c
Different letters
represent a significant difference among groups; p<0.01. △,Labre
○,Labre-in-chocolate;
powder;
□,NTM003-in-chocolate;
◇ ,NTM003 powder.
Fig. 3. Comparison of tolerance against simulated digestive juice in vitro between Labre-in-chocolate and several commercial probiotic foods. Each value is the mean±SD (n=3). The values of Protect C at after 0.5, 1, and 2 h were not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b ,c
Different letters represent a
significant difference among groups; p<0.01. ○,Labre-in-chocolate; △, Product B; □, Product C; ◇ , Product D.
Fig.
4.
Effect
of
fresh
and
freeze -dried
cream
on
tolerance
of
Labre-in-chocolate against simulated digestive juice. Each value is the mean±SD (n=3). The data, which subtract the values of 0. 5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a,b
Different letters represent a significant difference
between groups; p<0.01. ○,Labre-in-chocolate;
△,Labre-in-chocolate
Labre-in-chocolate with freeze-dried cream.
15
with
fresh
cream;
□,
Viable bacteria (Log cfu/ml)
7 6
a
a
a
5 4
b
3
b b
2
b
1
b Fig.1
0 0
16
1 Time (h)
2
Viable bacteria (Log cfu/ml)
7 6 5
a
a
a
a
a
a
4 b
3
b
b
b
c
2 1 0
0
1 Time (h)
Fig.2 17
2
Viable bacteria (Log cfu/ml )
7 6
a b c
5
a b c
4 3 2 1
0 0
1
2
Time (h) Fig.3 18
Variable bacteria (Log cfu/ml)
7 a
6 5
a
a a
a a
b
b
b
4 3 2
1 0
0
1 Time (h) 19
2
Fig.4
Ta b l e
1.
Comparison
of
the
nucleoside-transforming
activity
between Labre powder and Labre-in-chocolate after treatment with digestive juice or 0.5% NaCl Purine base
produceda
Guanine (mM) Hypoxanthi ne (mM) a
Labre powder
Labre-in-chocolate
Treatme
Treatme
Residu
Treatme
Treatme
Residu
nt with
nt with
al
nt with
nt with
al
0.85%
digestiv
ratioc
0.85%
digestiv
ratioc
NaCl
e juce
(%)
NaCl
e juce
(%)
0.208 ±
0.004 ±
2.0 ±
0.446 ±
0.320 ±
74.8 ±
0.035
0.004
1.7
0.083
0.159
39.6*d
0.489 ±
trb:<0.0
trb:<0.
1.316 ±
0.838 ±
65.4 ±
0.088
01
1
0.173
0.364
30.8
Labre powder and Labre-in-chocolate treated with simulated
digestive juice
or 0.85% NaCl
were used
for
guanosine and
inosine transforming reactions as described in materials and methods. Guanine and hypoxanthine produced from guanosine a n d i n o s i n e , r e s p e c t i v e l y, w e r e m e a s u r e d .
b
Tr a c e i s e x p r e s s e d
"tr" which means under the detection limits of the method. c
Residual
ratio
of
nucleoside-transforming
activity
after
treatment is calculated by dividing the value obtained with digestive
juice-treated
NaCl-treated sample.
d
sample
by
that
obtained
with
Differences of residual ratio are assessed
using Student’s t- test; *p < 0 .05. (n=3).
20
E a c h Va l u e i s t h e m e a n ± S D
Highlights: Chocolate processing enhanced toleranc e of probiotic bacteria against gastric acid.
Graphical abstract
21
PII: DOI: Reference:
www.elsevier.com/locate/bab
S1878-8181(15)00119-X http://dx.doi.org/10.1016/j.bcab.2015.09.001 BCAB298
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 14 July 2015 Revised date: 4 September 2015 Accepted date: 8 September 2015 Cite this article as: Yasunori Yonejima, Keiko Hisa, Marina Kawaguchi, Hiroaki Ashitani, Toshiyuki Koyama, Yoko Usamikrank, Nayumi Kishida, Shigenobu Kishino and Jun Ogawa, Lactic acid bacteria-containing chocolate as a practical probiotic Product with increased acid tolerance, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2015.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Lactic Acid Bacteria-Containing Chocolate as a Practical Probiotic Product with Increased Acid Tolerance
Author names and affiliations Yasunori Yonejima a , Keiko Hisa a , Marina Kawaguchi a , Hiroaki Ashitani b , Toshiyuki Koyama b , Yoko Usamikrank b , Nayumi Kishida c , Shigenobu Kishino c and Jun Ogawa c *
a
Research and Development Department, Nitto Pharmaceutical
Industries, Ltd., 35-3 Minamibiraki, Kamiueno-cho, Muko 617-0006, Japan b
Central Laboratory, LOTTE Co., Ltd., 3-1-1 Numakage Minami-ku,
Saitama 336-8601, Japan c
Division of Applied Life Science, Graduate School of Agriculture ,
Kyoto University, Kyoto 606-8502, Japan
* To whom correspondence should be addressed . Fax: +81-75-753-6113; E-mail: [email protected]
Abstract This study shows an evaluation of the method to improve acid tolerance of probiotic bacteria aimed at enhancement of the probiotic effects in the intestinal canal. In the tolerance test of simulated digestive juice in vitro, the viable rate of Lactobacillus brevis subsp. coagulans (Labre) processed with
chocolate
(Labre-in-chocolate)
is
significantly
increased,
approximately one hundred times higher, compared to those of freeze -dried Labre powder and Labre contained in beverage. This pro tective effect 1
depends on the content of water in chocolate for the probiotics processing. Differences in tolerance of metabolic activity were investigated with Labre-in-chocolate and freeze-dried Labre powder, both treated with simulated
digestive
Labre-in-chocolate
juice
remained
in
vitro.
significantly
The
enzyme
even
though
activity that
of
of the
freeze-dried Labre powder was inactivated. These results indicate that chocolate processing is an effective method making probiotics to be delivered to the intestine in a viable condition and to be e ffective for host health.
Key words: Lactobacillus brevis subsp. coagulans; Lactobacillus brevis NTM003; tolerance test of simulated digestive juice ; chocolate
1. Introduction
A bacterium that provides specific health benefits to the human body is called a probiotic, which is expected to have various benefits for health maintenance when consumed as a food component or supplement. Lactobacillus brevis subsp. coagulans called Labre is a lactic acid bacterium that belongs to probiotics. Labre is effective to improve the immune system through inducement of interferon-α production and enhancement of natural killer (NK) cell activity (Kishi et al., 1996; U.S.P.5662900). In other cases, it has been reported that Labre has various benefits for the human body such as early int ervention in irritable bowel syndrome (Murakami et al., 2012), activation the availability of medical herb flavonoid s (Sakurama et al., 2014). Moreover, there is a report that Labre could reduce the risk of infection in children 2
during influenza season (Waki et al., 2014). As discussed above, the effectiveness of probiotics such as Labre is now widely accepted by consumers, and this may play a part in the growth element of the functional food market. Lactic acid bacteria, however, are damaged by gastrointestinal stresses in the case of oral intake, and the viable rate is dramatically decreased in the intestine. It is expected to deliver live bacteria into the intestine to maintain the activities of the probiotics themselves or to protect their metabolisms generating effective compounds. Therefore, a live-bacteria-containing pharmaceutical preparation is coated with an enteric coating agent to prevent the pharmaceutical from being exposed to gastric acid aimed at enhancement of the probiotic effect s in the intestine. Lactic acid bacteria are usually taken with yogurts and beverages, but they are easily exposed to gastric acid. In this case, the probiotic effect s may not be expressed adequately in the intestine. The aim of this study is to establish the practical methods to deliver bacteria to the intestine in a viable condition in the form of general food through the verification of increased acid tolerance of Labre, which is coated
with
chocolate
against
simulated
digestive
juice
in
vitro.
Furthermore, we confirmed sustained enzyme activity in the lactic acid bacteria-in-chocolate after the treatment with simulated digestive juice with nucleoside-hydrolyzing activity as an indicator.
2. Materials and methods
2.1. Lactic acid bacteria Lactobacillus brevis subsp. coagulans (Labre) FERM BP-4693 used in this study is isolated from suguki zuke (a regional pickle of Japan made from a 3
turnip-like vegetable) in Kyoto, Japan, and the probiotic powder was produced at Nitto Pharmaceutical Industries, Ltd. (Kyoto, Japan). Labre was incubated at 30℃ for 24-48 h in a growth medium mainly composed of 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L meat extract. The culture supernatant was removed by centrifugation . The resultant bacterial cells were freeze-dried and used as Labre powder. Lactobacillus brevis NTM003 (NTM003) NITE BP-1634 used in this study is isolated from nanohanazuke (a regional pickle of Japan made from the flower of cole) in Kyoto, Japan, and the probiotic powd er was produced at Nitto Pharmaceutical Industries, Ltd.. NTM003 was incubated at 37℃ for 24-48 h in a growth medium mainly composed of 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, and 10 g/L meat extract. The culture supernatant was removed by centrifugation . The resultant bacterial cells were freeze-dried and used as NTM003 powder. Commercial probiotics used in this study are as follows: Product A is a probiotic beverage that contains Lactobacillus brevis. Product B is a probiotic beverage that contains Lactobacillus casei. Product C is a probiotic yogurt that contains some kind of lactic acid bacteria and is certified as a Food for Specified Health Uses in Japan. Product D is a world-wide consumed probiotic yogurt that contains some kinds of lactic acid bacteria.
2.2. Preparation of Labre-in-chocolate and NTM003-in-chocolate Labre or NTM003 powder was added to melted chocolate, and then this highly concentrated lactic acid bacteria-in-chocolate was taken into tempered chocolate, keeping the temperature around 32℃ . This chocolate liquid was poured into a mold, cooled, and used as probiotics-in-chocolate. Final concentration of Labre or NTM003 powder in the chocolate was 0.5% 4
(w/w). The used chocolate was a commercial chocolate (LOTTE Co., Ltd. Saitama, Japan) composed of 30% (w/w) cocoa components (cocoa mass and cocoa butter), 20% (w/w) milk component, 9% (w/w) vegetable fat component, 40% (w/w) sugar component, and 0.5% (w/w) emuls ifier and natural vanilla flavoring components.
2.3. Tolerance of lactic acid bacteria against simulated digestive juice in vitro A simulated digestive juice was made as described previously (Azuma et al., 2001) with modifications. Briefly, Lactobacilli MRS broth (Becton Dickinson, and Company, NJ, USA) with 0.04% (w/v) pepsin (Wako, Osaka, Japan)
was
adjusted
to
pH
2.5
using
HCl.
Four
gram
of
each
probiotics-in-chocolate were added to 300 ml of simulated digestive juice and shaken at 37℃ for a few hours at 70 rpm; the liquid surface was gently shaken by the revolutions per minute. Then the total viable count of probiotics was analyzed after 0.5 h, 1 h, and 2 h incubations in simulated digestive juice by sampling 1 ml of the solution, dil uting adequately, and plating on MRS broth plates with 1.5% (w/v) agar. For the treatment of probiotics powders or commercial probiotics products, the initial viable count
before
the
treatment
was
adjusted
as
many
as
it
of
probiotics-in-chocolate.
2.4. Probiotics-in-chocolate with fresh cream or freeze -dried cream Probiotics-in-chocolate with fresh cream was made as follows. Boiled fresh cream (20 g) was added to chopped chocolate (LOTTE Co., Ltd., Saitama, Japan) (50 g), and melted by heating at 40 ℃ . Then 0.5% (w/w) probiotics powder was added and mixed quickly. This chocolat e liquid was poured into a tray and cooled at 4℃ for 0.5 h. 5
Probiotics-in-chocolate with freeze-dried cream was made as follows. A chopped chocolate (50 g) was melted by heating at 40℃ . Then freeze-dried cream (10.4 g) and 0.5% (w/w) probiotics powder was added and mixed quickly. This chocolate liquid was poured into a tray and cooled at 4 ℃ for 1 h.
2.5. Activity analysis of the enzyme converting purine nucleosides into purine bases The enzyme activity of probiotics converting purine nucleosides into purine bases was assayed as described previously (Tsuboi et al., 2012; J.P.5149305) with modifications. Briefly, 50 mg of probiotics powder or 1 g of probiotics-in-chocolate which contained 50 mg of probiotics powder after treatment with 0.85% NaCl or simulated digestive juice was washed twice with 0.85% NaCl. After washing, 4.0 ml of the nucleoside solutions containing 2 mM guanosine and 4 mM inosine in 0.1 M potassium phosphate buffer (pH 7.0) were added into the resulting probiotics precipitation. The mixture was incubated for 1 h at 37°C, and then the reaction was terminated by adding 0.5 ml of 2.0 N HCl. Guanine and hypoxanthine
in
the
reaction
mixtures
were
quantified
with
high-performance liquid chromatography (HPLC). The HPLC analysis was performed with a Shimadzu LC 10A -VP system (Shimadzu, Kyoto, Japan) equipped with a Cosmosil 5C18 -PAQ column (4.6 × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) at 40°C. 10 μl of the filtered supernatant of the reaction mixture was injected to HPLC. The mobile phase was 50 mM KH 2 PO 4 (pH 3.0). The flow rate was 1.0 ml/min. The effluents were monitored at 254 nm using diode array detector (SPD -M20A, Shimadzu Kyoto, Japan).
6
2.6. Statistics Each result is expressed as the mean ±standard deviation (SD). Tukey’s multiple comparison tests o r S t u d e n t ’ s t - t e s t w e r e u s e d t o c o m p a r e e a c h g r o u p . Statistical analyses were done with Excel Toukei 2010 (Social Survey Research Information, Tokyo, Japan).
3. Results
3.1 Tolerance of differently processed probiotics against simulated digestive juice The acid tolerances of various probiotics in different preparations, i.e., Labre powder, Labre-in-chocolate, and Labre-in-beverage (Product A), were evaluated.
Fig. 1 shows the tolerance of lactic acid bacteria against
simulated digestive juice in vitro. The viable count of Labre powder was not stable; it immediately decreased from 10 6 cfu/ml to 10 3 cfu/ml in 0.5 h, notably decreased to 10 cfu/ml in 1 h, and finally was non-detected (marker not shown) in 2 h after adding into simulated digestive juice.
The viable
count of Labre-in-beverage (Product A) was also not stable: it immediately decreased from 10 6 cfu/ml to 10 3 cfu/ml in 0.5 h, notably decreased to 10 cfu/ml in 1 h, and finally decreased to 10 cfu/ml in 2 h after adding to simulated digestive juice. However, the viable count of Labre-in-chocolate was more stable for at least 2 h, it decreased slightly to 10 5 cfu/ml in 2 h after adding to simulated digestive juice. These results showed that the viable rate of Labre-in-chocolate is significantly increased , by at least one hundred
times,
compared
to
those
of
the
Labre
powder
and
Labre-in-beverage.
3.2 Effects of chocolate processing on different Lactobacillus brevis strains 7
Viability-enhancing effects of chocolate processing were investigated with two representative Lactobacillus brevis strains.
Fig. 2 shows a
comparison of tolerance against simulated digestive juice in vitro between Lactobacillus brevis subsp. coagulans (Labre) and Lactobacillus brevis NTM003 (NTM003). The tolerance test of lactic acid bacteria against simulated digestive juice in vitro was conducted as stated above. The viable rate
of
the
Labre-in-chocolate
was
stable,
and
th at
of
the
NTM003-in-chocolate was also stable. However, the viable rate of Labre powder was lower than that of NTM003 powder after 1 h. These results showed that the viable rates in simulated digestive juice differ in each of lactic acid bacterial strains, but the tolerances against simulated digestive juice were improved by adding them to chocolate.
3.3 Comparison of tolerance between Labre -in-chocolate and several probiotic foods Fig. 3 shows a comparison of tolerance against simulated digestive juice in vitro between Labre-in-chocolate and several probiotic foods. The tolerance test of lactic acid bacteria against simulated digestive juice in vitro
was
conducted
as
stated
above.
The
viable
count
of
Labre-in-chocolate was stable for at least 2 h in simulated digestive juice. On the other hand, the viable count s of Product B and Product D decreased gradually, and the viable count of Product C de creased immediately and was not detected (marker not shown) after 0.5 h in simulated digestive juice.
3.4 Effects of fresh cream and freeze-dried cream on protective effects of chocolate Fig. 4 shows a comparison of tolerance against simulated digestive juice between Labre-in-chocolate added with fresh cream or freeze -dried cream. 8
The tolerance test of lactic acid b acteria against simulated digestive juice in vitro
was
conducted
as
stated
above.
The
viable
counts
of
Labre-in-chocolate and those of Labre-in-chocolate added with freeze-dried cream
were
stable
for
at
least
2
h.
But
the
viable
count
of
Labre-in-chocolate added with fresh cream showed different results: it immediately decreased from 10 6 cfu/ml to 10 4 cfu/ml in 0.5 h, and after that it was kept at the same level at least 2 h. These results showed that the viable rate of Labre significantly decreases, by about ten times, by addition of fresh cream but not freeze-dried cream. These results indicated that the water in fresh cream affected protecting effects of chocolate on probiotics.
3.5 Effects of chocolate processing on the stability of the enzyme in probiotics converting purine nucleosides to purine bases Table 1 shows a comparison of enzyme activity converting purine nucleosides to purine bases between Labre powder and Labre-in-chocolate after treatment with simulated digestive juice. Control experiments were carried out after treatment with 0.85% NaCl instead of simulated digestive juice. Reactions were carried out with 2 mM guanosine and 4 mM inosine as substrates. In the reaction with Labre powder after treated 2 h with 0.85% NaCl, generations of 0.208 mM guanine and 0.489 mM hypoxanthine derived from decomposition of the substrates were observed after the reaction, but no generations of guanine and hypoxanthine were observed in the reaction with Labre powder treated by simulated digestive juice 2 h. On the other hand, in the reaction with Labre-in-chocolate treated 2 h with 0.85% NaCl, generations of 0.446 mM guanine and 1.316 mM hypoxanthine were observed, and
generations of 0.320 mM guanine and 0.838 mM
hypoxanthine were observed in the reaction with Labre-in-chocolate treated 2
h
with
simulated
digestive
juice. 9
These
results
showed
that
Labre-in-chocolate retained the enzyme activity in 0.85% NaCl and also in simulated digestive juice, indicating that chocolate processing is useful to stabilize the enz yme activity in probiotics during gastric acid treatment.
4. Discussion A large number of health-promoting effects of probiotics have been reported, and it commonly works on multiple people, however, it is reasonable to postulate that the majority of probiotics do not colonize in the intestine (Sanders, 2011) but is capable of growing during passage through the gastrointestinal track. A large population of living probiotics may be beneficial to effect host health as transient bacteria. A variety of studies have been conducted to demonstrate the positive effects of probiotics. Currently, metabolites produced by probiotics attract considerable attention among biochemists. Short-chain fatty acids produced by probiotics can act as metabolic regulators of energ y uptake in adipose tissue (Kimura et al., 2013). Lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition (Kishino et al., 2013). It is essential to deliver bacteria in a viable form to expect the effects of metabolites and enzymatic activity. Probiotics are commonly available in foods such as yogurt, beverages, freeze-dried powders, and tablets, but probiotics in these foods are not protected against gastric acid. Regarding oral medicines in the for m of powder, tablets and capsules are protected against gastric acid by an antacid or enteric coating agent. If such protection methods are easily applied in the manufacturing process of general foods, people can consume probiotic s that are delivered to the intestine in a viable condition. In this study, we aimed to establish practical and efficient methods to enhance the tolerance of probiotics to gastric acid. We observed the 10
increased viable rate of probiotics by coating with chocolate against simulated digestive juice in vitro. The fasting gastric pH value is generally 1 to 2, and it increases to 4 to 5 after eating and drink ing. It takes about 2 h for digested material to pass from the stomach to the duodenum, hence we set the pH value as 2.5 and 2 h duration as the conditions of the test. In the tolerance test in vitro, the viable rate of Labre coated with chocolate (Labre-in-chocolate) is significantly increased , approximately one hundred times higher, compared to those of Labre powder and Labre-in-beverages. It is considered that chocolate coats the surface of Labre to hardly soluble digestive juice and protect s Labre from the stress of low pH. We compared the effect of acid tolerance between Labre and NTM003, which is primarily stronger than Labre. The result showed that Labre-in-chocolate has effectiveness equal to that of NTM003 against simulated digestive juice. This result indicates that Labre-in-chocolate exerts an increased stability against gastric acid regardless of inh erent strength. Moreover, Labre-in-chocolate shows better tolerance against simulated digestive juice than that of probiotic yogurts on the market. To investigate the effect of water content in chocolate on protection against simulated digestive juice, we compared the tolerance between Labre-in-chocolate added with fresh cream or freeze-dried cream, which contains 10% and just a few amount of water, respectively. The viable rate of Labre-in-chocolate added with fresh cream is lower than th at in normal Labre-in-chocolate and Labre-in-chocolate added with freeze-dried cream. This result indicated that protection against simulated digestive juice diminished by water, which decreased the water-repellent effects may be caused by cacao lipids in the chocolate. Furthermore, we compared the enzyme activity converting purine nucleosides
into
purine
bases 11
between
Labre
powder
and
Labre-in-chocolate after treatment with digestive juice. The enzyme activity contributes to inhibiting the absorption of purine bodies (Tsuboi et al., 2012; J.P.5149305) and reduces serum uric acid contents. The enzyme activity of Labre-in-chocolate remains considerable level to maintain its biochemical effects, even though the Labre powder was lost the enzymatic activity. These results indicated that the expression of enzyme activity of Labre depends on increased viability in intestine caused by chocolate processing. In conclusion, chocolate processing is an effective way to deliver probiotics in a viable form to the intestine. Lipid s in chocolate may protect probiotics, are degraded by lipase in the intestine, and then release probiotics into intestine as a viable form and make them effective to the host health. Further studies to clarify the mechanisms of the protection are required.
5. Conclusions It is essential to deliver live bacteria into the intestine to maintain the activities as of the probiotics. Acid stress in stomach is one of the main causes of deactivation of live bacteria. So, we evaluated the method to improve acid tolerance of probiotic bacteria by coating in with chocolate. Chocolate processing was found as an effective way to deliver probiotics in a viable form to the intestine. Probiotics-in-chocolate was found to be more stable against gastric acid treatment than probiotics powder and those in beverages or in yogurt.
Chocolate processing was also useful to keep the
enzymatic activities of the probiotics against gastric acid treatment.
We
hope that this method improve the qualities of the probiotics contributing to human health and welfare.
12
Acknowledgment This work was supported, in part, by Scientific Research Grants in Aid 23688011 (to S.K.), and 26660064, 23248014 (to J.O.).
Reference Azuma, Y., Ito, K., Sato, M., 2001. Simulated digestive juice tolerance and inhibitory effect on harmful intestinal bacteria of Lactobacillus gasseri NY0509 and Lactobacillus casei NY1301. Nippon Shokuhin Kagaku Kogaku Kaishi. 48 (9), 656-663. Kimura, I., Ozawa, K., Inoue, D., Imamura, T., Kimura, K., Maeda, T., Terasawa, K., Kashihara, D., Hirano, K., Tani, T., Takahashi, T., Miyauchi, S., Shioi, G., Inoue, H., Tsujimoto, G., 2013. The gut microbiota
suppresses
insulin -mediated
short-chain
fatty
receptor
acid
GPR43.
fat
accumulation
Nat.
Commun.
via 4,
the DOI:
10.1038/ncomms2852. Kishi, A., Uno, K., Matsubara, Y., Okuda, C., Kishida, T., 1996. Effect of the oral administration of Lactobacillus brevis subsp. coagulans on ineterferon-alpha producing capacity in humans. J. Am. Coll. Nutr. 15 (4), 408-412. Kishino, S., Takeuchi, M., Park, S.B., Hirata, A., Kitam ura, N., Kunisawa, J., Kiyono, H., Iwamoto, R., Isobe, Y., Arita, M., Arai, H., Ueda, K., Shima, J., Takahashi, S., Yokozeki, K., Shimizu, S., Ogawa, J., 2013. Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc. Natl. Acad. Sci. U. S. A. 110 (44), 17808-17813. Murakami, K., Habukawa, C., Nobuta, Y., Moriguchi, N., Takemura, T., 2012. The effect of Lactobacillus brevis KB290 against irritable bowel syndrome:
a
placebo-controlled 13
double-blind
crossover
trial.
Biopsychosoc. Med. 6 (16), DOI: 10.1186/1751 -0759-6-16. Sakurama, H., Kishino, S., Uchibori, Y., Yonejima, Y., Ashida, H., Kita, K., Takahashi, S., Ogawa, J., 2014. β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to gly coside hydrolase family 30. Appl. Microbiol. Biotechnol. 98 (9), 4021-4032. Sanders, M.E., 2011. Impact of probiotics on colonizing microbiota of the gut. J. Clin. Gastroenterol. 45 , S115-S119. Tsuboi, Y., Kaneko, N., Sato, A., Lactic acid bacterium havin g effect of lowering blood uric acid level, J . Pat. No. 5149305. Tsunataro K., 1997. Method of Increasing interferon production in humans. U.S. Pat. No. 5662900. Waki, N., Matsumoto, M., Fukui, Y., Suganuma, H., 2014. Effects of probiotic Lactobacillus brevis KB290 on incidence of influenza infection among schoolchildren: an open -label pilot study. Lett. Appl. Microbiol. 59, 565-571.
Figure legends Fig. 1. Comparison of tolerance against simulated digestive juice in vitro between Labre powder, Labre-in-chocolate, and Labre-in-beverage (Product A). Each value is the mean±SD (n=3). The value of Labre powder at after 2 h was not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b
Different letters represent a
significant difference between groups; p<0.01. ○, Labre powder; △, Labre-in-chocolate; □, Labre-in-beverage (Product A).
Fig. 2. Comparison of tolerance against simulated digestive juice in vitro 14
between L. brevis strains. Each value is the mean±SD (n=3). The value of Labre powder at after 2 h was not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b , c
Different letters
represent a significant difference among groups; p<0.01. △,Labre
○,Labre-in-chocolate;
powder;
□,NTM003-in-chocolate;
◇ ,NTM003 powder.
Fig. 3. Comparison of tolerance against simulated digestive juice in vitro between Labre-in-chocolate and several commercial probiotic foods. Each value is the mean±SD (n=3). The values of Protect C at after 0.5, 1, and 2 h were not detected (marker not shown). The data, which subtract the values of 0.5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a ,b ,c
Different letters represent a
significant difference among groups; p<0.01. ○,Labre-in-chocolate; △, Product B; □, Product C; ◇ , Product D.
Fig.
4.
Effect
of
fresh
and
freeze -dried
cream
on
tolerance
of
Labre-in-chocolate against simulated digestive juice. Each value is the mean±SD (n=3). The data, which subtract the values of 0. 5, 1, or 2 h from that of the initial value (0 h), were assessed using Tukey’s multiple comparison tests.
a,b
Different letters represent a significant difference
between groups; p<0.01. ○,Labre-in-chocolate;
△,Labre-in-chocolate
Labre-in-chocolate with freeze-dried cream.
15
with
fresh
cream;
□,
Viable bacteria (Log cfu/ml)
7 6
a
a
a
5 4
b
3
b b
2
b
1
b Fig.1
0 0
16
1 Time (h)
2
Viable bacteria (Log cfu/ml)
7 6 5
a
a
a
a
a
a
4 b
3
b
b
b
c
2 1 0
0
1 Time (h)
Fig.2 17
2
Viable bacteria (Log cfu/ml )
7 6
a b c
5
a b c
4 3 2 1
0 0
1
2
Time (h) Fig.3 18
Variable bacteria (Log cfu/ml)
7 a
6 5
a
a a
a a
b
b
b
4 3 2
1 0
0
1 Time (h) 19
2
Fig.4
Ta b l e
1.
Comparison
of
the
nucleoside-transforming
activity
between Labre powder and Labre-in-chocolate after treatment with digestive juice or 0.5% NaCl Purine base
produceda
Guanine (mM) Hypoxanthi ne (mM) a
Labre powder
Labre-in-chocolate
Treatme
Treatme
Residu
Treatme
Treatme
Residu
nt with
nt with
al
nt with
nt with
al
0.85%
digestiv
ratioc
0.85%
digestiv
ratioc
NaCl
e juce
(%)
NaCl
e juce
(%)
0.208 ±
0.004 ±
2.0 ±
0.446 ±
0.320 ±
74.8 ±
0.035
0.004
1.7
0.083
0.159
39.6*d
0.489 ±
trb:<0.0
trb:<0.
1.316 ±
0.838 ±
65.4 ±
0.088
01
1
0.173
0.364
30.8
Labre powder and Labre-in-chocolate treated with simulated
digestive juice
or 0.85% NaCl
were used
for
guanosine and
inosine transforming reactions as described in materials and methods. Guanine and hypoxanthine produced from guanosine a n d i n o s i n e , r e s p e c t i v e l y, w e r e m e a s u r e d .
b
Tr a c e i s e x p r e s s e d
"tr" which means under the detection limits of the method. c
Residual
ratio
of
nucleoside-transforming
activity
after
treatment is calculated by dividing the value obtained with digestive
juice-treated
NaCl-treated sample.
d
sample
by
that
obtained
with
Differences of residual ratio are assessed
using Student’s t- test; *p < 0 .05. (n=3).
20
E a c h Va l u e i s t h e m e a n ± S D
Highlights: Chocolate processing enhanced toleranc e of probiotic bacteria against gastric acid.
Graphical abstract
21