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Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus
Accepted Manuscript Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus Jyun-Kai Jhan, Wei-Fen Chang, Pei-Ming Wang, Su-Tze Chou, Yun-Chin Chung PII:
S0023-6438(15)00261-3
DOI:
10.1016/j.lwt.2015.03.107
Reference:
YFSTL 4582
To appear in:
LWT - Food Science and Technology
Received Date: 5 September 2014 Revised Date:
24 March 2015
Accepted Date: 30 March 2015
Please cite this article as: Jhan, J.-K., Chang, W.-F., Wang, P.-M., Chou, S.-T., Chung, Y.-C., Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus, LWT - Food Science and Technology (2015), doi: 10.1016/ j.lwt.2015.03.107. 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 proof before it is published in its final 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.
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Production of fermented red beans with multiple bioactivities using
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co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp.
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bulgaricus
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Jyun-Kai Jhan, Wei-Fen Chang, Pei-Ming Wang, Su-Tze Chou and Yun-Chin
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Chung*
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Department of Food and Nutrition, Providence University, Shalu, Taichung 43301,
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Republic of China (Taiwan)
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Running Title: Microbially fermented red beans with multiple bioactivities
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The authors are affiliated with the Department of Food and Nutrition, Providence
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University,
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Tel:886-4-26328001,
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[email protected]. Address inquiries to Dr. Y. C. Chung.
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Chungchi
Shalu,
15345.
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Fax:
Taichung
43301,
Taiwan.
886-4-26530027.
e-mail:
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Abstract
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Red beans exhibit many biofunctions, including the stimulation of intestinal motility,
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improvement of anemia and elimination of edema. This study was conducted to
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evaluate the functional properties of microbially fermented red beans produced
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under different fermentation conditions and to establish the optimum
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fermentation conditions for the production of fermented red beans with
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multiple biofunctions. The optimum fermentation conditions were the fermentation
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of red beans by a co-culture of Bacillus subtilis and Lactobacillus bulgaricus in the
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presence of 1% glucose, incubated at 30℃ for 120 h and stirred every 24 h.
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Compared with unfermented red beans, red beans fermented under the optimum
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conditions contained a higher concentration of antioxidant substances, including total
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phenolics, anthocyanin, flavonoids and vitamins C and E. The results of tests for
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DPPH-radical scavenging, ferrous ion chelation and reducing power implied a high
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antioxidant content. Fermented red beans exhibited nattokinase activity and contained
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a significant amount of potential probiotics.
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Keywords：red bean, Bacillus subtilis, Lactobacillus delbrueckii subsp. bulgaricus,
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antioxidant activity, fibrinolytic activity
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Abbreviations: LAB, Lactic acid bacteria; t-PA, tissue plasminogen activator;
GABA, γ-aminobutyric acid; TPC, total phenolic content; TAC, total anthocyanin content;
TFC,
total
flavonoid
content;
NB,
nutrient
broth;
DPPH,
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α-α-diphenyl-β-pricryl-hydrazyl; FU, fibrinolytic activity;
α-Toc, α-Tocopherol;
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Trolox,
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2,2’-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid; CFU, colony-forming unit;
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AOAC, Association of Official Agricultural Chemists; IC50, half maximal
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
2
acid;
ABTS,
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inhibitory concentration; HPLC, high-performance liquid chromatography; S.D.,
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standard deviation; ANOVA, analysis of variance.
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1. Introduction Beans contain considerable amounts of phenolic compounds (Drumm et al.,
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1990; Srisuma et al., 1989) that have varying degrees of antioxidant activity, and
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the concentration of phenolic compounds in beans can be increased during the
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fermentation process (Muralami et al., 1984). The traditional Asian fermented soy
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foods such as miso, natto and tempeh have been found to exhibit remarkably
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stronger antioxidant activities than unfermented steamed soybeans (Berghofer et al.,
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1998; Esaki et al., 1994; Sheih et al., 2000). The red bean (Phaseolusradiatus L.
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var. Aurea) is a leguminous seed and is mainly used as a popular ingredient in
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oriental desserts. Unlike soy beans, red beans are seldom fermented. In fact, red
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beans contain many nutrients, such as carbohydrates, proteins, vitamins, minerals,
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fibers and saponin (Hoshikawa, 1985). In Chinese folk medication, red beans are
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commonly used for the treatment of constipation, anemia and edema.
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Many GRAS strains convert raw materials into desirable fermentation products.
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All fermented foods have aroma and flavor characteristics resulting from the
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fermentation process. In some instances, bioactive components are generated
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during the fermentation process. Lactic acid bacteria (LAB) are the best known of
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the beneficial strains used to ferment food products. The probiotic effects of LAB
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include the inhibition of pathogenic species (Berggren et al., 1993; Bernet et al.,
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1994), the strengthening of the body’s immune system (Berggren et al. 2011), the reduction of colon cancer (Thirabunyanon and Hongwittayakorn, 2013), anti-obesity (Tsai et al., 2014), the modulation of immune responses (Lee et al.,
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2011) and the decrease of serum cholesterol levels (Du Toit et al., 1998; Tahri et al.,
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1995). In addition to the probiotic role of L. bulgaricus, the antioxidant activity of
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this strain was demonstrated (Saide and Gilliland, 2005). The potential probiotic
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role of L. bulgaricus is still an interesting issue. Other studies demonstrated the 4
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probiotic effects of Bacillus subtilis, such as the antimicrobial activities against
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Salmonella Enteritidis (Thirabunyanon and Thongwittaya, 2012) and Vibrio
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anguillarum (Touraki et al., 2012), the modulation of the gut microbiota, the
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activation of non-specific immunity in shrimp (Zhang et al., 2011), and the
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improvement of calf immune functions (Sun et al., 2010).
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Natto is a traditional Japanese food product prepared by fermenting soybeans
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with B. subtilis. During natto processing, nattokinase is generated by the starter (B.
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subtilis) and causes the fibrinolytic activity of natto. To prevent thrombosis and
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other related diseases, daily intake of fibrinolytic enzymes from food sources is
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recommended. Recently, Kamiya’s group (Kamiya et al., 2010) proposed an
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antithrombotic mechanism for nattokinase according to results obtained by
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evaluating the effect of nattokinase on Carrageenan-induced tail thrombosis in a rat
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model. Kamiya et al. (2010) assumed that nattokinase has three anti-thrombosis
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functions, including the conversion of plasminogen to plasmin, the activation of
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t-PA (which also causes the transformation of plasminogen into plasmin) and
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finally, the degradation of fibrin by the fibrinolytic activity of plasmin together
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with nattokinase. In our previous studies, red beans replaced soybeans in
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fermentation with B. subtilis (the product was called natto-red beans). The 50%
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ethanolic extracts of non-fermented or B. subtilis fermented red beans showed
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antioxidant activities. The 50% ethanolic extract of B. subtilis-fermented red beans
was more effective than the non-fermented extract in raising antioxidant levels in
liver tissue (Chou et al., 2008). Furthermore, a fibrinolytic enzyme, a subtilisin-like
serine protease, was purified from natto-red beans (Chang et al., 2012).
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The purpose of the present paper was to produce a novel food with multiple
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bioactivities. Based on the biofunctions of natto and LAB, we used a co-culture of
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B. subtilis and Lactobacillus delbrueckii sp bulgaricus to produce fermented red 5
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beans with multiple biofunctions.
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2.1. Chemicals
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NB and MRS were purchased from HIMEDIA (LBS, India). DPPH, α-Toc, potassium ferricyanide, nisin (from Lactococus lactis),Trolox and ABTS were
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purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals
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were of reagent grade or higher purity.
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2.2. Preparation of starter cultures
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Bacillus subtilis (BCRC 14716) and Lactobacillus delbrueckii sp. bulgaricus
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(BCRC 14008) were purchased from the Bioresource Collection and Research
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Center (BCRC) at the Food Industry Research and Development Institution,
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Hsinchu, Taiwan. B. subtilis and L. bulgaricus were inoculated into NB and MRS
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broth, respectively. Frozen cultures of B. subtilis 6.65 log CFU mL-1 and L.
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bulgaricus 8.11 log CFU mL-1 were activated twice in 100 mL of medium (1: 9;
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v/v) in a 250-mL flask at 37
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without shaking for L. bulgaricus.
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for 4 h, with shaking at 250 rpm for B. subtilis or
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2.3. Inoculation method Two methods were applied to inoculate fermentation starts.
First, B.
subtilis (9.5 log CFU g-1) and L. bulgaricus (9.5 log CFU g-1) were simultaneously
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inoculated into steamed red beans, then incubated in a stainless steel tray at 37℃
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for 120 h.
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A second fermentation was initiated by inoculating L. bulgaricus after B. 6
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subtilis was allowed to grow for 48 h.
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2.4. Fermentation Red beans (Phaseolus radiatus L. var. Aurea, Kaohsiung #9) were obtained
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from the Tainan District Agriculture Improvement Station, Taiwan. The beans (150
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g) were soaked in 150 mL of dd H2O for 8 h. Solid-state fermentation was
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performed by inoculating a 1/10 (culture/red bean, v/w) starter (9.5 log CFU mL-1)
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into steamed (121°C for 1 h) and cooled red beans and then incubating under
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different fermentation conditions. Subsequently, the microbially fermented red
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beans were lyophilized and ground into powder.
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2.5. Fermentation conditions
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Five fermentation parameters were evaluated: (1) the addition of glucose,
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lactose or sucrose (1%, W/W) to steamed red beans, (2) fermentation in a cap-tight
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roller bottle (9.5 cm (D) × 23.0 cm (H), 1 turn/min) or in a stainless steel tray
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(28.0 cm (L) × 20.0 cm (W) × 5.5 cm (H)) covered with aluminum foil, (3)
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incubation in a temperature controlled fermentation room at 30 or 37℃, (4) with or
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without stirring the substrates every 24 h with a sterile spatulas, and (5) incubation
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for 0-120 h. Effects of the growth stage of starters on the biofunctions of fermented
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red beans were determined as well.
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2.6. Extracts of fermented red beans Both water extract and ethanolic extract were prepared. One gram of
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fermented red beans was suspended in 10 mL of dd H2O or 50% ethanol, and the
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mixture was stirred for 45 min. The precipitate was removed by centrifugation at
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8000×g for 10 min and the supernatant was filter-sterilized (filter pore size 0.45
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um). 7
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2.7. Microbial counts Fermented red beans (3 g) were homogenized by vortexing vigorously in a 50
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mL centrifuge tube containing 27 mL dd H2O. For B. subtilis, 0.1 mL of a serially
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diluted sample was spread on a nutrient agar plate and plates were incubated at
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for 24 h.
. For L. bulgaricus, 1.0 mL of serially diluted sample was poured on a 20 mL
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MRS agar plate containing 1x10-4 IU mL-1 nisin. Viable L. bulgaricus were
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counted after the plate was incubated at 37
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2.8. Compositions determination
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for 48 h.
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Analysis of protein (AOAC 984.13), lipid (AOAC 954.02), moisture (AOAC
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AOAC 934.01), ash (AOAC 942.05) and fiber (AOAC 962.09) content was
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performed according to the standard AOAC method (1997).
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2.9. Measurements of antioxidant activities
The DPPH radical-scavenging activities of the extracts were measured according
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to the method of Yamaguchi et al. (1998). The reducing power and Fe2+-chelation
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activity were determined according to Oyaizu and Deiezak (1986), respectively. The
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half maximal inhibitory concentration (IC50) was calculated as the antioxidant
concentration required for providing 50% of the antioxidant activity.
2.10. Determination of fibrinolytic activity
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Two assays, the fibrin plate assay and nattokinase activity, were performed to
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determine the fibrinolytic activity of the fermented red beans according to Astrup 8
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and Müllertz (1952) and Deepak et al. (2008), respectively. One unit of fibrinolytic
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activity (FU) is defined as the amount of enzyme required to produce an increase in
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absorbance equal to 0.01 in one min at 275 nm (Deepak et al., 2008).
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2.11. Measurements of vitamin B12, C, E and GABA concentration
Levels of vitamins B12, C. and E in the tested samples were measured using
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HPLC with UV (371 nm), electrochemical and UV (300 nm) detectors according to
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the methods of Quesada-Chanto et al. (1998), Albrecht and Schafer (1990) and
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Nierenberg and Nann (1992), respectively.
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The GABA content was determined by HPLC with a C18 column according to Kim et al. (2009).
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2.12. Determinations of total phenolics, flavonoids and the anthocyanin content Total phenolic content was analyzed using the Folin-Ciocalteu reagent method
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(Sato et al., 1996) with gallic acid as the standard for the calibration curve, and the
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total phenolic content was expressed as mg gallic acid equivalents per gram of tested
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extract. The total flavonoid content of the samples was determined using a modified
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colorimetric method with rutin as the standard (Zhishen et al., 1999). The
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anthocyanin content of the extracts was analyzed according to the method of
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Padmavati et al. (1997).
2.13. Statistical Analysis
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To established the optimum fermentation conditions, single experiment with
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three measurements was performed for each condition, such as sugars, fermenters,
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fermentation times and mixing.
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Three independent experiments were conducted to evaluate the bioactivities of 9
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fermented red beans under optimum fermentation conditions. Analysis of variance was performed by ANOVA procedures using SPSS 10.0
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software (Spss Inc. Chicago, IL, USA). Duncan’s new multiple-range test was used to
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determine the differences among means. When only two groups were compared,
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mean values were compared by Student’s t-test or analysis of variance. A significance
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level of 5% was adopted for all comparisons.
3. Results and Discussion
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3.1. Optimum conditions for solid state fermentation
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The optimum fermentation conditions were established for red bean
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fermentation by testing different conditions, such as different fermentation sugars
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(1% glucose, 1% sucrose or 1% lactose), different fermenters (roller bottles or
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stainless steel trays), fermentation times (0,12, 24, 48, 72, 96 or 120 h),
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temperatures (30℃ or 37℃) and mixing. We expected to establish optimal
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fermentation conditions by evaluating characteristics of fermented red beans such
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as the biomass of B. subtilis and L. bulgaricus, fibrinolytic activity and the DPPH
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scavenging effect. Functional properties of red beans fermented under optimal
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conditions were determined, including the approximate composition of fermented
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products, viable counts of B. subtilis and L. bulgaricus, pH, antimicrobial activity,
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fibrinolytic activity, antioxidant activities (including DPPH radical scavenging,
reducing power and ferrous ion chelation), total polyphenols, anthocyanin and
flavonoid contents, vitamin content (B12, C, E) and GABA (γ-aminobutyric acid) content.
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3.1.1. Effects of sugars To evaluate the effect of sugars on the growth of each starter culture, 1% 10
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glucose, sucrose or lactose was mixed with steamed red beans. The fermentation
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was performed in a stainless steel tray at 37℃ for 96 h. The growth of both
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cultures reached a maximum after incubation for 12 h, and red beans with 1%
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glucose showed the highest growth of B. subtilis (8.26 log CFU g-1). Compared to
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the control (no added sugar), the three tested sugars did not cause significant
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differences in the growth of L. bulgaricus (p>0.05). After incubated for 12 h, the
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maximum viable count of L. bulgaricus was in the range of 8.64-8.90 log CFU g-1.
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Addition of 1% glucose stimulated the growth of B. subtilis but had no effect on
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that of L. bulgaricus; therefore, fermentation substrates (red beans) were mixed
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with 1% glucose (w/w) for the rest of the study.
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3.1.2. Inoculation method
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Fig. 1A. shows the pH change and viable counts for B. subtilis and L.
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bulgaricus during fermentation when two strains were simultaneously inoculated
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into steamed red beans. The maximum number of viable cells (9.55 log CFU g-1) of
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B. subtilis was detected after incubation for 24 h. L. bulgaricus had a peak growth
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(8.94 log CFU g-1) after 72 h of incubation. The pH of the red beans increased
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during fermentation, indicating that protein degradation exceeded sugar
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fermentation.
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L. bulgaricus was inoculated after B. subtilis was allowed to grow for 48 h
(Fig. 1B). The pH changes and growth of B. subtilis during the fermentation period were similar to that of the two starter cultures that were simultaneously inoculated.
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L. bulgaricus was inoculated two days after the growth of B. subtilis because we
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expected L. bulgaricus to grow fast in an anaerobic environment, which was
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created once oxygen was depleted by the aerobic growth of B. subtilis. Surprisingly,
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L. bulgaricus did not grow and died quickly under these conditions. The pH 11
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changes and growth of B. subtilis during the fermentation period were similar to
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those when the two starter cultures were simultaneously inoculated. We suspected
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that substances produced by B. subtilis might be toxic to L. bulgaricus. We did not try to inoculate B. subtilis after L. bulgaricus was pre-inoculated.
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In our pretest, we found that pH of the fermented red beans dropped from 6.75 to
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5.30 when the steamed red beans were incubated with L. bulgaricus for two days,
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and the resultant (acidified red beans) inhibited the growth of B. subtilis (data not
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shown).
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Our previous study showed that B. subtilis-fermented red beans contained a
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fibrinolytic
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fibrinolytic/caseinolytic activity (using fibrin/casein as a substrate) was comparable
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to that of commercial nattokinase (Chang et al. 2012). Therefore, the fermented red
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beans were assessed for their fibrinolytic activity to evaluate the effect of the
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inoculation method on the production of the fibrinolytic enzyme. The red bean
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sample with the highest fibrinolytic activity was obtained at 96 h after
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simultaneous inoculation of the two cultures (Table 1).
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serine
protease,
and
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For rest of the study, two starter cultures were inoculated in steamed red
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beans simultaneously.
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enzyme,
3.2.3. Fermenter
Upon incubation in a roller bottle at 37℃ for 120 h, both B. subtilis (5.86 log
CFU g-1) and L. bulgaricus (6.4 log CFU g-1) grew as well as they did in pure
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cultures, reaching 8.52 log CFU g-1 and 7.47 log CFU g-1, respectively; however,
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the biomass of B. subtilis decreased from 5.94 to 3.40 log CFU g-1 under co-culture
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conditions (data not shown). When oxygen was depleted by B. subtilis, the aerobic
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growth of B. subtilis was inhibited in the tightly capped roller bottles; conversely, 12
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the growth of L. bulgaricus increased (from 6.21 to 8.91 log CFU g-1) in the
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co-culture system (data not shown). Neither pure nor co-cultures produced a clear
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zone on the fibrin plate assay. According to these results, a roller bottle is not a
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suitable fermenter for co-cultures of B. subtilis and L. bulgaricus.
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3.2.4. Effects of stirring on the growth of the starter cultures
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We assumed that stirring the red beans (every 24 h) during the fermentation
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process might favor the aerobic growth of B. subtilis but be detrimental to the
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anaerobic growth of L. bulgaricus. The viable cells of B. subtilis increased from
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5.86 log CFU g-1 to 9.63 log CFU g-1 and 10.55 log CFU g-1 without and with
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stirring, respectively, after fermentation at 37℃ for 120 h (Fig. 2A). The viable
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cells of L. bulgaricus increased from 6.34 log CFU g-1 to 8.09 log CFU g-1 and
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8.01 log CFU g-1 without and with stirring, respectively, after fermentation at 37℃
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for 120 h (Fig. 2B). Stirring of the red beans significantly increased the growth of
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B. subtilis (p<0.05), but did not affect the growth of L. bulgaricus.
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3.2.5. Fermentation temperature
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Because the optimum growth temperature for B. subtilis (30℃) was not the
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same as that of L. bulgaricus (37℃), the red beans were incubated at either 30℃ or
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37℃. After fermentation at 30℃ for 120 h, viable cells of B. subtilis increased from 5.87 log CFU g-1 to 10.60 log CFU g-1 and 10.57 log CFU g-1 without and
with stirring, respectively (Fig. 2A). Compared to fermentation at 37℃ (Fig 2A),
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fermentation at 30℃ did not increase the growth of B. subtilis when the
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fermentation substrates were stirred. The viable cells of L. bulgaricus increased
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from 6.15 log CFU g-1 to 6.21 log CFU g-1 and 7.00 log CFU g-1 without and with
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stirring, respectively, when fermentation proceeded at 30℃ for 120 h (Fig. 2B). 13
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On the other hand, 30℃ incubation was superior to 37℃ incubation for the
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production of the fibrinolytic enzyme based on the observation that the highest
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fibrinolytic activity was obtained for 30℃/120 h/stirred red bean fermentation
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(Table 2). Because fibrinolytic activity was considered to be the most desirable
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bioactivity in fermented red beans, 30℃ was selected as the optimum fermentation
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temperature. The 30℃-fermented red bean culture contained a high biomass of
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both bacteria, even though it had fewer viable cells of L. bulgaricus than that for
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red beans fermented at 37℃.
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3.2.6. Fermentation time
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In addition to fibrinolytic activity, antioxidant activity was assumed to be a
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potential bioactivity of fermented red beans. The water extract of 30℃-fermented
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red beans was assayed for DPPH radical-scavenging activity during the
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fermentation process. The lowest IC50 was detected in the 30℃/120 h/stirring -
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fermented red beans inoculated simultaneously with the two starters (Table 3), and
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this culture had the highest fibrinolytic activity.
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3.3. Multiple biofunctions of red beans fermented under optimum conditions According the results mentioned above, the optimum fermentation conditions
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were (1) the addition of 1% glucose to the steamed red beans, (2) fermentation by a
co-culture of B. subtilis and L. bulgaricus in a stainless steel tray at 30℃ for 120 h,
and (3) stirring of the beans every 24 h. The following parameters were assessed to
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evaluate the quality of the final product: biomass, pH, changes in the composition
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of the red beans, and antioxidant and fibrinolytic activities.
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3.3.1. Biomass and pH 14
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The initial counts of B. subtilis and L. bulgaricus were 5.94 log CFU g-1 and
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6.27 log CFU g-1, respectively. After fermentation for 120 h, the viable cells of B.
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subtilis and L. bulgaricus increased to 9.03 log CFU g-1 and 8.12 log CFU g-1,
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respectively (data not shown). A significant amount of active probiotics in the
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fermentation product indicated that this product had potential gastrointestinal
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functionality.
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The starting pH of the fermentation substrate was 6.83, which decreased to
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6.09 after fermentation for 120 h. Red beans were acidified by the lactic acid or
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other organic acids produced by L. bulgaricus. During fermentation, the pH of the
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fermented substrate was maintained in a suitable range for production of the
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fibrinolytic enzyme as well as for the growth of B. subtilis. The maximal
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fibrinolytic activity produced by B. subtilis was at pH 6.52 (Ashipala and He,
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2008), and optimal pH for the growth of L. bulgaricus was pH 5.8-6.0 (Rault et al.,
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2009). However, the pH of L. bulgaricus fermented red beans was not as low as
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that of L. bulgaricus fermented dairy products (around pH 4.5), possibly because
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nutrients for L. bulgaricus were limited.
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Even though viable counts of L. bulgaricus increased and the pH decreased
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during fermentation, the fermented red beans did not show antimicrobial activity
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toward foodborne bacteria, such as Escherichia coli, Listeria monocytogenes,
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Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enterica subsp. Enterica, Staphylococcus aureus, Enterobacter aerogenes and Bacillus cereus (data not shown).
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3.3.2. Changes in the compositions of the red beans
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Compared to unfermented red beans, total fiber and protein content was
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increased and total carbohydrate and lipid content was decreased (p<0.05) in red 15
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beans fermented under optimum conditions (Table 4). Notably, the decreased
364
carbohydrate content indicated that, in addition to the 1% glucose added at the
365
beginning of the fermentation that served as a nutrient for bacterial growth,
366
approximately 43% of the carbohydrates in the red beans were decomposed during
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the fermentation process. The content of vitamins C and E increased dramatically.
368
However, neither vitamin B12 nor GABA was detected in either the unfermented
369
and fermented samples.
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Kim et al. (2009) fermented black raspberry juice with Lactobacillus brevis
371
GABA100, and GABA (27.6 mg mL-1) was detected in the fermented juice.
372
However, neither B. subtilis nor L. bulgaricus produced GABA under our
373
fermentation conditions.
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3.3.3. Antioxidant activities
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Red beans fermented with a co-culture of B. subtilis and L. bulgaricus
377
exhibited antioxidant activities, including DPPH radical-scavenging activity,
378
reducing power, and Fe2+-chelation activity. Ethanol extracts showed greater
379
antioxidant activity than water extracts (Table 5).
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The potent antioxidants in red beans were phenolic substances, anthocyanin and
381
flavonoids. The antioxidants increased during the fermentation process (Table 5), and
382 383 384
AC C
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the ethanol extract had a higher level of antioxidants than the water extract, which
explained the higher antioxidant activities. Similar results were reported in
heonggukjang, Bacillus subtilis fermented soybeans, with the total flavanol and
385
phenol content and the DPPH radical scavenging activity increasing during
386
fermentation (Cho et al., 2011).
387 388
3.3.4. Fibrinolytic activity 16
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There was no detectable fibrinolytic activity in unfermented red beans. After
390
fermentation, nattokinase activity (28.21±0.56 FU g-1) was detected and a fibrin
391
plate assay also showed an obvious clear zone (21.97 mm), indicating that fibrin
392
was hydrolyzed by the water extract of fermented red beans (data not shown).
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389
393 394
4. Conclusions
This study established the optimum fermentation conditions for the production of
396
fermented red beans with multiple biofunctions. The fermentation conditions studied
397
included the addition of different sugars (1% glucose, 1% sucrose or 1% lactose),
398
different fermenters (roller bottles or stainless steel tray), fermentation times (0, 12,
399
24, 48, 72, 96 or 120 h), temperatures (30℃ or 37℃) and stirring. Optimal
400
fermentation conditions were expected to be determined by measuring the
401
characteristics of fermented red beans such as the biomass of B. subtilis and L.
402
bulgaricus, fibrinolytic activity and DPPH scavenging activity. The optimum
403
fermentation conditions were obtained with a co-culture of B. subtilis (9.5 log CFU
404
g-1) and L. bulgaricus (9.5 log CFU g-1 ) in the addition of 1% glucose, incubated in a
405
stainless steel tray (28.0 cm (L) × 20.0 cm (W) × 5.5 cm (H)) covered with aluminum
406
foil, stirring the red beans every 24 h with a sterile spatulas, and incubated in a
407
temperature controlled fermentation room (30℃) for 120 h. Under these conditions,
409 410
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AC C
408
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the fermented red beans had the lowest IC50 value for DPPH scavenging and the greatest fibrinolytic activities. The biofunctions of red beans fermented under optimal conditions were highly viable probiotics (B. subtilis and L. bulgaricus), high
411
antioxidant properties, and fibrinolytic activity. Compared with unfermented red
412
beans, red beans fermented with B. subtilis and L. bulgaricus had higher polyphenol,
413
anthocyanin, flavonoid, vitamin C and E content and a higher fibrinolytic activity. In
414
this study, a novel red bean product with multi-bioactivities, such as probiotic 17
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potential, antioxidants and clot dissolving potential, was produced by fermenting red
416
beans with co-cultures of B. subtilis and L. bulgaricus. To maintain the function of
417
fibrinolytic activity and the viability of fermentation strains (L. bulgaricus and B.
418
subtilis), capsulated products is most desired.
419
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415
Acknowledgments
421
This research was supported by the National Science Council, Taiwan
422
(NSC97-2313-B-126-003-MY3). Its financial support is greatly appreciated.
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420
423
Fig. 1. Biomass and pH changes in the fermented red beans using co-cultures of B. subtilis and L. bulgaricus during fermentation. Fermentation conditions：37℃ without stirring and fermented in a
427 428 429 430 431
tray fermenter. Each value is the mean ± SD (single experiment with three measurements single experiment with three measurements ). A: two starters inoculated simultaneously; B: L. bulgaricus inoculated after B. subtilis was allowed to grow for 48 h. －●－ B. subtilis, －○－ L. bulgaricus, －△－ pH.
432 433 434 435 436 437 438
Fig. 2. Effects of incubation temperature and stirring on the growth of starters in the fermented red beans using co-cultures of B. subtilis and L. bulgaricus during fermentation processes. Each value is the mean ± SD (single experiment with three measurements single experiment with three measurements ).A: growth of B. subtilis; B: growth of L. bulgaricus. －●－ 30℃ without stirring, －○－ 30℃ with stirring, －▼－ 37℃ without stirring, －△－ 37℃
440 441
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439
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with stirring.
18
Table 1. Fibrinolytic activity of fermented red beans*. Starter strain
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Fermentation time (h)
Ad
24
48 ABa
0
17.00 ± 0.02
B. subtilis and L. bulgaricus L. bulgaricus followed with B. subtilis**
0Ac 0Ac
17.73 ± 0.14Aab 15.96 ± 0.02Bb
13.66 ± 0.06
96 Aa
16.76 ± 0.06
120 Cb
15.03 ± 0.07
*Fermentation was performed at 37℃ without stirring the fermented substrates during the fermentation process. Each value (mean± SD,
single experiment with three
measurements ) is the diameter (mm) of the colorless zone using the fibrin plate assay. ** L. bulgaricus was inoculated after B. subtilis was allowed to grow for 48 h.
a-c
Means in the same column followed by different letters are significantly different (p＜0.05). One-way ANOVA, Duncan’s multiple range test, P > 0.05.
TE D
A-C
AC C
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Means in the same row followed by different letters are significantly different (p＜0.05). One-way ANOVA, Duncan’s multiple range test, P > 0.05.
19
17.40 ± 0.05Aa
16.93 ± 0.01Ab 17.16 ± 0.10Ab 19.50 ± 0.04Aa 18.16 ± 0.18Aab 17.23 ± 0.03Aa 16.17 ± 0.03Ab 17.43 ± 0.08Ba 16.33 ± 0.08Ab
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B. subtilis
72
Bc
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0
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Fermentation time (h) 0
24
48
72
without stirring
0.00b
16.33 ± 0.12a
16.67 ± 0.08a
stirring*** 37℃
0.00c
17.93 ± 0.17b
17.03 ± 0.10b
without stirring stirring
0.00d 0.00c
14.60 ± 0.05c 12.33 ± 0.08*b
14.50 ± 0.09c 18.00 ± 0.10a
96
120
18.40 ± 0.28a
17.80 ± 0.10a
16.23 ± 0.03a
19.30 ± 0.30b
22.67 ± 0.10*a 23.83 ± 0.20*a
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30℃
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Table 2. Effect of stirring on the fibrinolytic activity of fermented red beans using co-cultures of B. subtilis and L. bulgaricus **.
16.70 ± 0.17ab 15.77 ± 0.07bc 18.33 ± 0.15a 18.30 ± 0.05*a
17.83 ± 0.12a 18.33 ± 0.06a
**Fermentation was performed at 30 or 37℃with/without stirring the fermented substrates during the fermentation process. Each value (mean± SD,
single experiment with three measurements) is the diameter (mm) of the colorless zone using the fibrin plate assay. *
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***Stirring the fermentation substrates every 12 h during the fermentation process.
Significantly different from the control group (without stirring). One-way ANOVA, Student’s t test, P > 0.05.
a-c
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Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
20
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Table 3. DPPH radical scavenging ability expressed by the half maximal inhibitory concentration (IC50, mg mL-1) for the 30℃fermented red beans. Fermentation time (h) 48 b
24.15±0.08 19.37±0.08 27.14±0.08*a 14.07±0.08*e
Each value is the mean ± SD (single
72 c
17.37±0.08 18.67±0.08b
96
c
120
17.45±0.08 15.17±0.08 16.87±0.08c 14.93±0.08*d 15.68±0.08*c 11.28±0.08*f
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without stirring stirring
24 a
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0
d
experiment with three measurements).
*
Significantly different from the control group (without stirring). One-way ANOVA, Student’s t test, P > 0.05.
a-f
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Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
21
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Table 4. Approximate composition and vitamins and γ- aminobutyric acid (GABA) contents of unfermented red beans and red beans fermented under optimal conditions. Optimum fermented red beans
Crude fiber (%)
4.34 ± 0.18
4.85 ± 0.22*
Crude lipid (%) Crude protein (%) Crude ash (%) Carbohydrate (%) Vitamin B12 (mg g-1) Vitamin C (mg g-1) Vitamin E (mg g-1) γ- aminobutyric acid (GABA)
1.12 ± 0.05* 22.68 ± 0.33 0.78 ± 0.05 12.82 ± 0.2* ND 5.41 ± 0.12 0.08 ± 0.06 ND
0.17 ± 0.22 24.27 ± 0.41* 0.79 ± 0.03 6.65 ± 0.21 ND 192.12 ± 0.09* 0.38 ± 0.16* ND
Data are the mean ± SD (three *
independent experiments).
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ND: not detected.
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Unfermented red beans
AC C
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Significantly higher than the other group. One-way ANOVA, Student’s t test, P > 0.05.
22
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Table 5. Antioxidant content and antioxidant activities of different extracts from unfermented red beans and red beans fermented under optimum conditions. Unfermented red beans
Optimum fermented red beans
50% Ethanol
1% HCl/ methanol
Water
50% Ethanol
1% HCl/ methanol
Total phenols (mg gallic acid/ g sample)
2.30±0.02d
2.59±0.01d
ND
3.25±0.04b
3.63±0.03a
ND
Total anthocyanins (µmole/ g sample)
ND
ND
0.03±0.06b
ND
ND
0.04±0.01a
Total flavonoids (mg rutin/ g sample)
ND
2.40±0.02b
ND
ND
2.64±0.07a
ND
IC50 of DPPH scavenging ability (mg mL-1)
84.55±0.75d
56.00±2.99c
ND
22.43±0.97b
16.64±0.08a
ND
Reducing power (A700 at 0.06 g mL-1)
0.65±0.00c
0.69±0.02c
0.46±0.02d
2.66±0.08b
3.00±0.00a
ND
ND
ND
ND
＞1000
75.32±27.72
ND
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EP
AC C
IC50 of Fe2+ chelating ability (mg mL-1)
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Water
23
ND: not detected. Each value is the mean ± SD (three
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Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
AC C
a-d
independent experiments ).
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1. We produced a novel fermented red beans with multi-bioactivities. 2. Co-culture of B. subtilis and L. bulgaricus was applied.
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significant amount of viable probiotics.
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S0023-6438(15)00261-3
DOI:
10.1016/j.lwt.2015.03.107
Reference:
YFSTL 4582
To appear in:
LWT - Food Science and Technology
Received Date: 5 September 2014 Revised Date:
24 March 2015
Accepted Date: 30 March 2015
Please cite this article as: Jhan, J.-K., Chang, W.-F., Wang, P.-M., Chou, S.-T., Chung, Y.-C., Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus, LWT - Food Science and Technology (2015), doi: 10.1016/ j.lwt.2015.03.107. 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 proof before it is published in its final 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.
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Production of fermented red beans with multiple bioactivities using
2
co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp.
3
bulgaricus
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Jyun-Kai Jhan, Wei-Fen Chang, Pei-Ming Wang, Su-Tze Chou and Yun-Chin
6
Chung*
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Department of Food and Nutrition, Providence University, Shalu, Taichung 43301,
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Republic of China (Taiwan)
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Running Title: Microbially fermented red beans with multiple bioactivities
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The authors are affiliated with the Department of Food and Nutrition, Providence
16
University,
17
Tel:886-4-26328001,
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[email protected]. Address inquiries to Dr. Y. C. Chung.
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Chungchi
Shalu,
15345.
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Ext.
Rd.,
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Fax:
Taichung
43301,
Taiwan.
886-4-26530027.
e-mail:
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Abstract
23
Red beans exhibit many biofunctions, including the stimulation of intestinal motility,
24
improvement of anemia and elimination of edema. This study was conducted to
25
evaluate the functional properties of microbially fermented red beans produced
26
under different fermentation conditions and to establish the optimum
27
fermentation conditions for the production of fermented red beans with
28
multiple biofunctions. The optimum fermentation conditions were the fermentation
29
of red beans by a co-culture of Bacillus subtilis and Lactobacillus bulgaricus in the
30
presence of 1% glucose, incubated at 30℃ for 120 h and stirred every 24 h.
31
Compared with unfermented red beans, red beans fermented under the optimum
32
conditions contained a higher concentration of antioxidant substances, including total
33
phenolics, anthocyanin, flavonoids and vitamins C and E. The results of tests for
34
DPPH-radical scavenging, ferrous ion chelation and reducing power implied a high
35
antioxidant content. Fermented red beans exhibited nattokinase activity and contained
36
a significant amount of potential probiotics.
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Keywords：red bean, Bacillus subtilis, Lactobacillus delbrueckii subsp. bulgaricus,
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antioxidant activity, fibrinolytic activity
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Abbreviations: LAB, Lactic acid bacteria; t-PA, tissue plasminogen activator;
GABA, γ-aminobutyric acid; TPC, total phenolic content; TAC, total anthocyanin content;
TFC,
total
flavonoid
content;
NB,
nutrient
broth;
DPPH,
44
α-α-diphenyl-β-pricryl-hydrazyl; FU, fibrinolytic activity;
α-Toc, α-Tocopherol;
45
Trolox,
46
2,2’-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid; CFU, colony-forming unit;
47
AOAC, Association of Official Agricultural Chemists; IC50, half maximal
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
2
acid;
ABTS,
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inhibitory concentration; HPLC, high-performance liquid chromatography; S.D.,
49
standard deviation; ANOVA, analysis of variance.
50
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1. Introduction Beans contain considerable amounts of phenolic compounds (Drumm et al.,
55
1990; Srisuma et al., 1989) that have varying degrees of antioxidant activity, and
56
the concentration of phenolic compounds in beans can be increased during the
57
fermentation process (Muralami et al., 1984). The traditional Asian fermented soy
58
foods such as miso, natto and tempeh have been found to exhibit remarkably
59
stronger antioxidant activities than unfermented steamed soybeans (Berghofer et al.,
60
1998; Esaki et al., 1994; Sheih et al., 2000). The red bean (Phaseolusradiatus L.
61
var. Aurea) is a leguminous seed and is mainly used as a popular ingredient in
62
oriental desserts. Unlike soy beans, red beans are seldom fermented. In fact, red
63
beans contain many nutrients, such as carbohydrates, proteins, vitamins, minerals,
64
fibers and saponin (Hoshikawa, 1985). In Chinese folk medication, red beans are
65
commonly used for the treatment of constipation, anemia and edema.
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Many GRAS strains convert raw materials into desirable fermentation products.
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All fermented foods have aroma and flavor characteristics resulting from the
68
fermentation process. In some instances, bioactive components are generated
69
during the fermentation process. Lactic acid bacteria (LAB) are the best known of
70
the beneficial strains used to ferment food products. The probiotic effects of LAB
71
include the inhibition of pathogenic species (Berggren et al., 1993; Bernet et al.,
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1994), the strengthening of the body’s immune system (Berggren et al. 2011), the reduction of colon cancer (Thirabunyanon and Hongwittayakorn, 2013), anti-obesity (Tsai et al., 2014), the modulation of immune responses (Lee et al.,
75
2011) and the decrease of serum cholesterol levels (Du Toit et al., 1998; Tahri et al.,
76
1995). In addition to the probiotic role of L. bulgaricus, the antioxidant activity of
77
this strain was demonstrated (Saide and Gilliland, 2005). The potential probiotic
78
role of L. bulgaricus is still an interesting issue. Other studies demonstrated the 4
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probiotic effects of Bacillus subtilis, such as the antimicrobial activities against
80
Salmonella Enteritidis (Thirabunyanon and Thongwittaya, 2012) and Vibrio
81
anguillarum (Touraki et al., 2012), the modulation of the gut microbiota, the
82
activation of non-specific immunity in shrimp (Zhang et al., 2011), and the
83
improvement of calf immune functions (Sun et al., 2010).
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Natto is a traditional Japanese food product prepared by fermenting soybeans
85
with B. subtilis. During natto processing, nattokinase is generated by the starter (B.
86
subtilis) and causes the fibrinolytic activity of natto. To prevent thrombosis and
87
other related diseases, daily intake of fibrinolytic enzymes from food sources is
88
recommended. Recently, Kamiya’s group (Kamiya et al., 2010) proposed an
89
antithrombotic mechanism for nattokinase according to results obtained by
90
evaluating the effect of nattokinase on Carrageenan-induced tail thrombosis in a rat
91
model. Kamiya et al. (2010) assumed that nattokinase has three anti-thrombosis
92
functions, including the conversion of plasminogen to plasmin, the activation of
93
t-PA (which also causes the transformation of plasminogen into plasmin) and
94
finally, the degradation of fibrin by the fibrinolytic activity of plasmin together
95
with nattokinase. In our previous studies, red beans replaced soybeans in
96
fermentation with B. subtilis (the product was called natto-red beans). The 50%
97
ethanolic extracts of non-fermented or B. subtilis fermented red beans showed
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antioxidant activities. The 50% ethanolic extract of B. subtilis-fermented red beans
was more effective than the non-fermented extract in raising antioxidant levels in
liver tissue (Chou et al., 2008). Furthermore, a fibrinolytic enzyme, a subtilisin-like
serine protease, was purified from natto-red beans (Chang et al., 2012).
102
The purpose of the present paper was to produce a novel food with multiple
103
bioactivities. Based on the biofunctions of natto and LAB, we used a co-culture of
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B. subtilis and Lactobacillus delbrueckii sp bulgaricus to produce fermented red 5
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beans with multiple biofunctions.
106 2. Materials and Methods
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2.1. Chemicals
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NB and MRS were purchased from HIMEDIA (LBS, India). DPPH, α-Toc, potassium ferricyanide, nisin (from Lactococus lactis),Trolox and ABTS were
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purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals
112
were of reagent grade or higher purity.
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2.2. Preparation of starter cultures
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Bacillus subtilis (BCRC 14716) and Lactobacillus delbrueckii sp. bulgaricus
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(BCRC 14008) were purchased from the Bioresource Collection and Research
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Center (BCRC) at the Food Industry Research and Development Institution,
118
Hsinchu, Taiwan. B. subtilis and L. bulgaricus were inoculated into NB and MRS
119
broth, respectively. Frozen cultures of B. subtilis 6.65 log CFU mL-1 and L.
120
bulgaricus 8.11 log CFU mL-1 were activated twice in 100 mL of medium (1: 9;
121
v/v) in a 250-mL flask at 37
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without shaking for L. bulgaricus.
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2.3. Inoculation method Two methods were applied to inoculate fermentation starts.
First, B.
subtilis (9.5 log CFU g-1) and L. bulgaricus (9.5 log CFU g-1) were simultaneously
127
inoculated into steamed red beans, then incubated in a stainless steel tray at 37℃
128
for 120 h.
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A second fermentation was initiated by inoculating L. bulgaricus after B. 6
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subtilis was allowed to grow for 48 h.
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2.4. Fermentation Red beans (Phaseolus radiatus L. var. Aurea, Kaohsiung #9) were obtained
133
from the Tainan District Agriculture Improvement Station, Taiwan. The beans (150
134
g) were soaked in 150 mL of dd H2O for 8 h. Solid-state fermentation was
135
performed by inoculating a 1/10 (culture/red bean, v/w) starter (9.5 log CFU mL-1)
136
into steamed (121°C for 1 h) and cooled red beans and then incubating under
137
different fermentation conditions. Subsequently, the microbially fermented red
138
beans were lyophilized and ground into powder.
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2.5. Fermentation conditions
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Five fermentation parameters were evaluated: (1) the addition of glucose,
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lactose or sucrose (1%, W/W) to steamed red beans, (2) fermentation in a cap-tight
143
roller bottle (9.5 cm (D) × 23.0 cm (H), 1 turn/min) or in a stainless steel tray
144
(28.0 cm (L) × 20.0 cm (W) × 5.5 cm (H)) covered with aluminum foil, (3)
145
incubation in a temperature controlled fermentation room at 30 or 37℃, (4) with or
146
without stirring the substrates every 24 h with a sterile spatulas, and (5) incubation
147
for 0-120 h. Effects of the growth stage of starters on the biofunctions of fermented
148
red beans were determined as well.
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2.6. Extracts of fermented red beans Both water extract and ethanolic extract were prepared. One gram of
152
fermented red beans was suspended in 10 mL of dd H2O or 50% ethanol, and the
153
mixture was stirred for 45 min. The precipitate was removed by centrifugation at
154
8000×g for 10 min and the supernatant was filter-sterilized (filter pore size 0.45
155
um). 7
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2.7. Microbial counts Fermented red beans (3 g) were homogenized by vortexing vigorously in a 50
159
mL centrifuge tube containing 27 mL dd H2O. For B. subtilis, 0.1 mL of a serially
160
diluted sample was spread on a nutrient agar plate and plates were incubated at
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37
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for 24 h.
. For L. bulgaricus, 1.0 mL of serially diluted sample was poured on a 20 mL
163
MRS agar plate containing 1x10-4 IU mL-1 nisin. Viable L. bulgaricus were
164
counted after the plate was incubated at 37
166
2.8. Compositions determination
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for 48 h.
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Analysis of protein (AOAC 984.13), lipid (AOAC 954.02), moisture (AOAC
168
AOAC 934.01), ash (AOAC 942.05) and fiber (AOAC 962.09) content was
169
performed according to the standard AOAC method (1997).
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2.9. Measurements of antioxidant activities
The DPPH radical-scavenging activities of the extracts were measured according
173
to the method of Yamaguchi et al. (1998). The reducing power and Fe2+-chelation
174
activity were determined according to Oyaizu and Deiezak (1986), respectively. The
176 177 178
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half maximal inhibitory concentration (IC50) was calculated as the antioxidant
concentration required for providing 50% of the antioxidant activity.
2.10. Determination of fibrinolytic activity
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Two assays, the fibrin plate assay and nattokinase activity, were performed to
180
determine the fibrinolytic activity of the fermented red beans according to Astrup 8
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and Müllertz (1952) and Deepak et al. (2008), respectively. One unit of fibrinolytic
182
activity (FU) is defined as the amount of enzyme required to produce an increase in
183
absorbance equal to 0.01 in one min at 275 nm (Deepak et al., 2008).
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2.11. Measurements of vitamin B12, C, E and GABA concentration
Levels of vitamins B12, C. and E in the tested samples were measured using
187
HPLC with UV (371 nm), electrochemical and UV (300 nm) detectors according to
188
the methods of Quesada-Chanto et al. (1998), Albrecht and Schafer (1990) and
189
Nierenberg and Nann (1992), respectively.
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The GABA content was determined by HPLC with a C18 column according to Kim et al. (2009).
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2.12. Determinations of total phenolics, flavonoids and the anthocyanin content Total phenolic content was analyzed using the Folin-Ciocalteu reagent method
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(Sato et al., 1996) with gallic acid as the standard for the calibration curve, and the
196
total phenolic content was expressed as mg gallic acid equivalents per gram of tested
197
extract. The total flavonoid content of the samples was determined using a modified
198
colorimetric method with rutin as the standard (Zhishen et al., 1999). The
199
anthocyanin content of the extracts was analyzed according to the method of
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Padmavati et al. (1997).
2.13. Statistical Analysis
203
To established the optimum fermentation conditions, single experiment with
204
three measurements was performed for each condition, such as sugars, fermenters,
205
fermentation times and mixing.
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Three independent experiments were conducted to evaluate the bioactivities of 9
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fermented red beans under optimum fermentation conditions. Analysis of variance was performed by ANOVA procedures using SPSS 10.0
209
software (Spss Inc. Chicago, IL, USA). Duncan’s new multiple-range test was used to
210
determine the differences among means. When only two groups were compared,
211
mean values were compared by Student’s t-test or analysis of variance. A significance
212
level of 5% was adopted for all comparisons.
3. Results and Discussion
215
3.1. Optimum conditions for solid state fermentation
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The optimum fermentation conditions were established for red bean
217
fermentation by testing different conditions, such as different fermentation sugars
218
(1% glucose, 1% sucrose or 1% lactose), different fermenters (roller bottles or
219
stainless steel trays), fermentation times (0,12, 24, 48, 72, 96 or 120 h),
220
temperatures (30℃ or 37℃) and mixing. We expected to establish optimal
221
fermentation conditions by evaluating characteristics of fermented red beans such
222
as the biomass of B. subtilis and L. bulgaricus, fibrinolytic activity and the DPPH
223
scavenging effect. Functional properties of red beans fermented under optimal
224
conditions were determined, including the approximate composition of fermented
225
products, viable counts of B. subtilis and L. bulgaricus, pH, antimicrobial activity,
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fibrinolytic activity, antioxidant activities (including DPPH radical scavenging,
reducing power and ferrous ion chelation), total polyphenols, anthocyanin and
flavonoid contents, vitamin content (B12, C, E) and GABA (γ-aminobutyric acid) content.
230 231 232
3.1.1. Effects of sugars To evaluate the effect of sugars on the growth of each starter culture, 1% 10
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glucose, sucrose or lactose was mixed with steamed red beans. The fermentation
234
was performed in a stainless steel tray at 37℃ for 96 h. The growth of both
235
cultures reached a maximum after incubation for 12 h, and red beans with 1%
236
glucose showed the highest growth of B. subtilis (8.26 log CFU g-1). Compared to
237
the control (no added sugar), the three tested sugars did not cause significant
238
differences in the growth of L. bulgaricus (p>0.05). After incubated for 12 h, the
239
maximum viable count of L. bulgaricus was in the range of 8.64-8.90 log CFU g-1.
240
Addition of 1% glucose stimulated the growth of B. subtilis but had no effect on
241
that of L. bulgaricus; therefore, fermentation substrates (red beans) were mixed
242
with 1% glucose (w/w) for the rest of the study.
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3.1.2. Inoculation method
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Fig. 1A. shows the pH change and viable counts for B. subtilis and L.
246
bulgaricus during fermentation when two strains were simultaneously inoculated
247
into steamed red beans. The maximum number of viable cells (9.55 log CFU g-1) of
248
B. subtilis was detected after incubation for 24 h. L. bulgaricus had a peak growth
249
(8.94 log CFU g-1) after 72 h of incubation. The pH of the red beans increased
250
during fermentation, indicating that protein degradation exceeded sugar
251
fermentation.
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L. bulgaricus was inoculated after B. subtilis was allowed to grow for 48 h
(Fig. 1B). The pH changes and growth of B. subtilis during the fermentation period were similar to that of the two starter cultures that were simultaneously inoculated.
255
L. bulgaricus was inoculated two days after the growth of B. subtilis because we
256
expected L. bulgaricus to grow fast in an anaerobic environment, which was
257
created once oxygen was depleted by the aerobic growth of B. subtilis. Surprisingly,
258
L. bulgaricus did not grow and died quickly under these conditions. The pH 11
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changes and growth of B. subtilis during the fermentation period were similar to
260
those when the two starter cultures were simultaneously inoculated. We suspected
261
that substances produced by B. subtilis might be toxic to L. bulgaricus. We did not try to inoculate B. subtilis after L. bulgaricus was pre-inoculated.
263
In our pretest, we found that pH of the fermented red beans dropped from 6.75 to
264
5.30 when the steamed red beans were incubated with L. bulgaricus for two days,
265
and the resultant (acidified red beans) inhibited the growth of B. subtilis (data not
266
shown).
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Our previous study showed that B. subtilis-fermented red beans contained a
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fibrinolytic
269
fibrinolytic/caseinolytic activity (using fibrin/casein as a substrate) was comparable
270
to that of commercial nattokinase (Chang et al. 2012). Therefore, the fermented red
271
beans were assessed for their fibrinolytic activity to evaluate the effect of the
272
inoculation method on the production of the fibrinolytic enzyme. The red bean
273
sample with the highest fibrinolytic activity was obtained at 96 h after
274
simultaneous inoculation of the two cultures (Table 1).
277 278 279 280
serine
protease,
and
its
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For rest of the study, two starter cultures were inoculated in steamed red
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beans simultaneously.
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enzyme,
3.2.3. Fermenter
Upon incubation in a roller bottle at 37℃ for 120 h, both B. subtilis (5.86 log
CFU g-1) and L. bulgaricus (6.4 log CFU g-1) grew as well as they did in pure
281
cultures, reaching 8.52 log CFU g-1 and 7.47 log CFU g-1, respectively; however,
282
the biomass of B. subtilis decreased from 5.94 to 3.40 log CFU g-1 under co-culture
283
conditions (data not shown). When oxygen was depleted by B. subtilis, the aerobic
284
growth of B. subtilis was inhibited in the tightly capped roller bottles; conversely, 12
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the growth of L. bulgaricus increased (from 6.21 to 8.91 log CFU g-1) in the
286
co-culture system (data not shown). Neither pure nor co-cultures produced a clear
287
zone on the fibrin plate assay. According to these results, a roller bottle is not a
288
suitable fermenter for co-cultures of B. subtilis and L. bulgaricus.
289 290
3.2.4. Effects of stirring on the growth of the starter cultures
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We assumed that stirring the red beans (every 24 h) during the fermentation
292
process might favor the aerobic growth of B. subtilis but be detrimental to the
293
anaerobic growth of L. bulgaricus. The viable cells of B. subtilis increased from
294
5.86 log CFU g-1 to 9.63 log CFU g-1 and 10.55 log CFU g-1 without and with
295
stirring, respectively, after fermentation at 37℃ for 120 h (Fig. 2A). The viable
296
cells of L. bulgaricus increased from 6.34 log CFU g-1 to 8.09 log CFU g-1 and
297
8.01 log CFU g-1 without and with stirring, respectively, after fermentation at 37℃
298
for 120 h (Fig. 2B). Stirring of the red beans significantly increased the growth of
299
B. subtilis (p<0.05), but did not affect the growth of L. bulgaricus.
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3.2.5. Fermentation temperature
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Because the optimum growth temperature for B. subtilis (30℃) was not the
303
same as that of L. bulgaricus (37℃), the red beans were incubated at either 30℃ or
304 305 306
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37℃. After fermentation at 30℃ for 120 h, viable cells of B. subtilis increased from 5.87 log CFU g-1 to 10.60 log CFU g-1 and 10.57 log CFU g-1 without and
with stirring, respectively (Fig. 2A). Compared to fermentation at 37℃ (Fig 2A),
307
fermentation at 30℃ did not increase the growth of B. subtilis when the
308
fermentation substrates were stirred. The viable cells of L. bulgaricus increased
309
from 6.15 log CFU g-1 to 6.21 log CFU g-1 and 7.00 log CFU g-1 without and with
310
stirring, respectively, when fermentation proceeded at 30℃ for 120 h (Fig. 2B). 13
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On the other hand, 30℃ incubation was superior to 37℃ incubation for the
312
production of the fibrinolytic enzyme based on the observation that the highest
313
fibrinolytic activity was obtained for 30℃/120 h/stirred red bean fermentation
314
(Table 2). Because fibrinolytic activity was considered to be the most desirable
315
bioactivity in fermented red beans, 30℃ was selected as the optimum fermentation
316
temperature. The 30℃-fermented red bean culture contained a high biomass of
317
both bacteria, even though it had fewer viable cells of L. bulgaricus than that for
318
red beans fermented at 37℃.
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3.2.6. Fermentation time
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In addition to fibrinolytic activity, antioxidant activity was assumed to be a
322
potential bioactivity of fermented red beans. The water extract of 30℃-fermented
323
red beans was assayed for DPPH radical-scavenging activity during the
324
fermentation process. The lowest IC50 was detected in the 30℃/120 h/stirring -
325
fermented red beans inoculated simultaneously with the two starters (Table 3), and
326
this culture had the highest fibrinolytic activity.
329 330 331 332
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3.3. Multiple biofunctions of red beans fermented under optimum conditions According the results mentioned above, the optimum fermentation conditions
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were (1) the addition of 1% glucose to the steamed red beans, (2) fermentation by a
co-culture of B. subtilis and L. bulgaricus in a stainless steel tray at 30℃ for 120 h,
and (3) stirring of the beans every 24 h. The following parameters were assessed to
333
evaluate the quality of the final product: biomass, pH, changes in the composition
334
of the red beans, and antioxidant and fibrinolytic activities.
335 336
3.3.1. Biomass and pH 14
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The initial counts of B. subtilis and L. bulgaricus were 5.94 log CFU g-1 and
338
6.27 log CFU g-1, respectively. After fermentation for 120 h, the viable cells of B.
339
subtilis and L. bulgaricus increased to 9.03 log CFU g-1 and 8.12 log CFU g-1,
340
respectively (data not shown). A significant amount of active probiotics in the
341
fermentation product indicated that this product had potential gastrointestinal
342
functionality.
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The starting pH of the fermentation substrate was 6.83, which decreased to
344
6.09 after fermentation for 120 h. Red beans were acidified by the lactic acid or
345
other organic acids produced by L. bulgaricus. During fermentation, the pH of the
346
fermented substrate was maintained in a suitable range for production of the
347
fibrinolytic enzyme as well as for the growth of B. subtilis. The maximal
348
fibrinolytic activity produced by B. subtilis was at pH 6.52 (Ashipala and He,
349
2008), and optimal pH for the growth of L. bulgaricus was pH 5.8-6.0 (Rault et al.,
350
2009). However, the pH of L. bulgaricus fermented red beans was not as low as
351
that of L. bulgaricus fermented dairy products (around pH 4.5), possibly because
352
nutrients for L. bulgaricus were limited.
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Even though viable counts of L. bulgaricus increased and the pH decreased
354
during fermentation, the fermented red beans did not show antimicrobial activity
355
toward foodborne bacteria, such as Escherichia coli, Listeria monocytogenes,
357 358
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Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enterica subsp. Enterica, Staphylococcus aureus, Enterobacter aerogenes and Bacillus cereus (data not shown).
359 360
3.3.2. Changes in the compositions of the red beans
361
Compared to unfermented red beans, total fiber and protein content was
362
increased and total carbohydrate and lipid content was decreased (p<0.05) in red 15
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beans fermented under optimum conditions (Table 4). Notably, the decreased
364
carbohydrate content indicated that, in addition to the 1% glucose added at the
365
beginning of the fermentation that served as a nutrient for bacterial growth,
366
approximately 43% of the carbohydrates in the red beans were decomposed during
367
the fermentation process. The content of vitamins C and E increased dramatically.
368
However, neither vitamin B12 nor GABA was detected in either the unfermented
369
and fermented samples.
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Kim et al. (2009) fermented black raspberry juice with Lactobacillus brevis
371
GABA100, and GABA (27.6 mg mL-1) was detected in the fermented juice.
372
However, neither B. subtilis nor L. bulgaricus produced GABA under our
373
fermentation conditions.
374 375
3.3.3. Antioxidant activities
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Red beans fermented with a co-culture of B. subtilis and L. bulgaricus
377
exhibited antioxidant activities, including DPPH radical-scavenging activity,
378
reducing power, and Fe2+-chelation activity. Ethanol extracts showed greater
379
antioxidant activity than water extracts (Table 5).
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The potent antioxidants in red beans were phenolic substances, anthocyanin and
381
flavonoids. The antioxidants increased during the fermentation process (Table 5), and
382 383 384
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the ethanol extract had a higher level of antioxidants than the water extract, which
explained the higher antioxidant activities. Similar results were reported in
heonggukjang, Bacillus subtilis fermented soybeans, with the total flavanol and
385
phenol content and the DPPH radical scavenging activity increasing during
386
fermentation (Cho et al., 2011).
387 388
3.3.4. Fibrinolytic activity 16
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There was no detectable fibrinolytic activity in unfermented red beans. After
390
fermentation, nattokinase activity (28.21±0.56 FU g-1) was detected and a fibrin
391
plate assay also showed an obvious clear zone (21.97 mm), indicating that fibrin
392
was hydrolyzed by the water extract of fermented red beans (data not shown).
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4. Conclusions
This study established the optimum fermentation conditions for the production of
396
fermented red beans with multiple biofunctions. The fermentation conditions studied
397
included the addition of different sugars (1% glucose, 1% sucrose or 1% lactose),
398
different fermenters (roller bottles or stainless steel tray), fermentation times (0, 12,
399
24, 48, 72, 96 or 120 h), temperatures (30℃ or 37℃) and stirring. Optimal
400
fermentation conditions were expected to be determined by measuring the
401
characteristics of fermented red beans such as the biomass of B. subtilis and L.
402
bulgaricus, fibrinolytic activity and DPPH scavenging activity. The optimum
403
fermentation conditions were obtained with a co-culture of B. subtilis (9.5 log CFU
404
g-1) and L. bulgaricus (9.5 log CFU g-1 ) in the addition of 1% glucose, incubated in a
405
stainless steel tray (28.0 cm (L) × 20.0 cm (W) × 5.5 cm (H)) covered with aluminum
406
foil, stirring the red beans every 24 h with a sterile spatulas, and incubated in a
407
temperature controlled fermentation room (30℃) for 120 h. Under these conditions,
409 410
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the fermented red beans had the lowest IC50 value for DPPH scavenging and the greatest fibrinolytic activities. The biofunctions of red beans fermented under optimal conditions were highly viable probiotics (B. subtilis and L. bulgaricus), high
411
antioxidant properties, and fibrinolytic activity. Compared with unfermented red
412
beans, red beans fermented with B. subtilis and L. bulgaricus had higher polyphenol,
413
anthocyanin, flavonoid, vitamin C and E content and a higher fibrinolytic activity. In
414
this study, a novel red bean product with multi-bioactivities, such as probiotic 17
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potential, antioxidants and clot dissolving potential, was produced by fermenting red
416
beans with co-cultures of B. subtilis and L. bulgaricus. To maintain the function of
417
fibrinolytic activity and the viability of fermentation strains (L. bulgaricus and B.
418
subtilis), capsulated products is most desired.
419
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Acknowledgments
421
This research was supported by the National Science Council, Taiwan
422
(NSC97-2313-B-126-003-MY3). Its financial support is greatly appreciated.
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Fig. 1. Biomass and pH changes in the fermented red beans using co-cultures of B. subtilis and L. bulgaricus during fermentation. Fermentation conditions：37℃ without stirring and fermented in a
427 428 429 430 431
tray fermenter. Each value is the mean ± SD (single experiment with three measurements single experiment with three measurements ). A: two starters inoculated simultaneously; B: L. bulgaricus inoculated after B. subtilis was allowed to grow for 48 h. －●－ B. subtilis, －○－ L. bulgaricus, －△－ pH.
432 433 434 435 436 437 438
Fig. 2. Effects of incubation temperature and stirring on the growth of starters in the fermented red beans using co-cultures of B. subtilis and L. bulgaricus during fermentation processes. Each value is the mean ± SD (single experiment with three measurements single experiment with three measurements ).A: growth of B. subtilis; B: growth of L. bulgaricus. －●－ 30℃ without stirring, －○－ 30℃ with stirring, －▼－ 37℃ without stirring, －△－ 37℃
440 441
TE D
EP
AC C
439
M AN U
424 425 426
with stirring.
18
Table 1. Fibrinolytic activity of fermented red beans*. Starter strain
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Fermentation time (h)
Ad
24
48 ABa
0
17.00 ± 0.02
B. subtilis and L. bulgaricus L. bulgaricus followed with B. subtilis**
0Ac 0Ac
17.73 ± 0.14Aab 15.96 ± 0.02Bb
13.66 ± 0.06
96 Aa
16.76 ± 0.06
120 Cb
15.03 ± 0.07
*Fermentation was performed at 37℃ without stirring the fermented substrates during the fermentation process. Each value (mean± SD,
single experiment with three
measurements ) is the diameter (mm) of the colorless zone using the fibrin plate assay. ** L. bulgaricus was inoculated after B. subtilis was allowed to grow for 48 h.
a-c
Means in the same column followed by different letters are significantly different (p＜0.05). One-way ANOVA, Duncan’s multiple range test, P > 0.05.
TE D
A-C
AC C
EP
Means in the same row followed by different letters are significantly different (p＜0.05). One-way ANOVA, Duncan’s multiple range test, P > 0.05.
19
17.40 ± 0.05Aa
16.93 ± 0.01Ab 17.16 ± 0.10Ab 19.50 ± 0.04Aa 18.16 ± 0.18Aab 17.23 ± 0.03Aa 16.17 ± 0.03Ab 17.43 ± 0.08Ba 16.33 ± 0.08Ab
M AN U
B. subtilis
72
Bc
SC
0
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Fermentation time (h) 0
24
48
72
without stirring
0.00b
16.33 ± 0.12a
16.67 ± 0.08a
stirring*** 37℃
0.00c
17.93 ± 0.17b
17.03 ± 0.10b
without stirring stirring
0.00d 0.00c
14.60 ± 0.05c 12.33 ± 0.08*b
14.50 ± 0.09c 18.00 ± 0.10a
96
120
18.40 ± 0.28a
17.80 ± 0.10a
16.23 ± 0.03a
19.30 ± 0.30b
22.67 ± 0.10*a 23.83 ± 0.20*a
M AN U
SC
30℃
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Table 2. Effect of stirring on the fibrinolytic activity of fermented red beans using co-cultures of B. subtilis and L. bulgaricus **.
16.70 ± 0.17ab 15.77 ± 0.07bc 18.33 ± 0.15a 18.30 ± 0.05*a
17.83 ± 0.12a 18.33 ± 0.06a
**Fermentation was performed at 30 or 37℃with/without stirring the fermented substrates during the fermentation process. Each value (mean± SD,
single experiment with three measurements) is the diameter (mm) of the colorless zone using the fibrin plate assay. *
TE D
***Stirring the fermentation substrates every 12 h during the fermentation process.
Significantly different from the control group (without stirring). One-way ANOVA, Student’s t test, P > 0.05.
a-c
AC C
EP
Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
20
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Table 3. DPPH radical scavenging ability expressed by the half maximal inhibitory concentration (IC50, mg mL-1) for the 30℃fermented red beans. Fermentation time (h) 48 b
24.15±0.08 19.37±0.08 27.14±0.08*a 14.07±0.08*e
Each value is the mean ± SD (single
72 c
17.37±0.08 18.67±0.08b
96
c
120
17.45±0.08 15.17±0.08 16.87±0.08c 14.93±0.08*d 15.68±0.08*c 11.28±0.08*f
M AN U
without stirring stirring
24 a
SC
0
d
experiment with three measurements).
*
Significantly different from the control group (without stirring). One-way ANOVA, Student’s t test, P > 0.05.
a-f
AC C
EP
TE D
Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
21
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Table 4. Approximate composition and vitamins and γ- aminobutyric acid (GABA) contents of unfermented red beans and red beans fermented under optimal conditions. Optimum fermented red beans
Crude fiber (%)
4.34 ± 0.18
4.85 ± 0.22*
Crude lipid (%) Crude protein (%) Crude ash (%) Carbohydrate (%) Vitamin B12 (mg g-1) Vitamin C (mg g-1) Vitamin E (mg g-1) γ- aminobutyric acid (GABA)
1.12 ± 0.05* 22.68 ± 0.33 0.78 ± 0.05 12.82 ± 0.2* ND 5.41 ± 0.12 0.08 ± 0.06 ND
0.17 ± 0.22 24.27 ± 0.41* 0.79 ± 0.03 6.65 ± 0.21 ND 192.12 ± 0.09* 0.38 ± 0.16* ND
Data are the mean ± SD (three *
independent experiments).
M AN U TE D
ND: not detected.
SC
Unfermented red beans
AC C
EP
Significantly higher than the other group. One-way ANOVA, Student’s t test, P > 0.05.
22
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Table 5. Antioxidant content and antioxidant activities of different extracts from unfermented red beans and red beans fermented under optimum conditions. Unfermented red beans
Optimum fermented red beans
50% Ethanol
1% HCl/ methanol
Water
50% Ethanol
1% HCl/ methanol
Total phenols (mg gallic acid/ g sample)
2.30±0.02d
2.59±0.01d
ND
3.25±0.04b
3.63±0.03a
ND
Total anthocyanins (µmole/ g sample)
ND
ND
0.03±0.06b
ND
ND
0.04±0.01a
Total flavonoids (mg rutin/ g sample)
ND
2.40±0.02b
ND
ND
2.64±0.07a
ND
IC50 of DPPH scavenging ability (mg mL-1)
84.55±0.75d
56.00±2.99c
ND
22.43±0.97b
16.64±0.08a
ND
Reducing power (A700 at 0.06 g mL-1)
0.65±0.00c
0.69±0.02c
0.46±0.02d
2.66±0.08b
3.00±0.00a
ND
ND
ND
ND
＞1000
75.32±27.72
ND
M AN U
TE D
EP
AC C
IC50 of Fe2+ chelating ability (mg mL-1)
SC
Water
23
ND: not detected. Each value is the mean ± SD (three
EP
TE D
M AN U
SC
Means in the same row followed by different letters are significantly different. One-way ANOVA, Duncan’s multiple range test, P > 0.05.
AC C
a-d
independent experiments ).
RI PT
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24
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AC C
EP
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12
10
9
8
8
6
7
4
6
5
0
20
40
60
80
100
120
M AN U
Fermentation Time (h) 12
10
B 10
9
8
8
6
7
4
2
0 0
20
TE D
Bacteria (log CFU/g)
SC
2
40
60
80
AC C
EP
Fermentation time (h)
Fig.1.
pH value
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10
1
6
5 100
120
pH value
Bacteria (log CFU/g)
A
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11
A 9
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B. subtilis (log CFU/g)
10
8
7
5 0
20
40
60
80
100
9.5
B 9.0 8.5
TE D
8.0 7.5 7.0 6.5 6.0 5.5
20
AC C
0
EP
L. Bulgaricus (log CFU/g)
120
M AN U
Fermentation time (h)
SC
6
40
60
80
100
Fermentation time (h)
Fig. 2.
2
120
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1. We produced a novel fermented red beans with multi-bioactivities. 2. Co-culture of B. subtilis and L. bulgaricus was applied.
AC C
EP
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M AN U
SC
significant amount of viable probiotics.
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3. Fermented red bean exhibited antioxidant activities, fibrinolytic activity and