Cracking Streptococcus thermophilus to stimulate the growth of the probiotic Lactobacillus casei in co-culture Chengjie Ma, Aimin Ma, Guangyu Gong, Zhenmin Liu, Zhengjun Wu, Benheng Guo, Zhengjun Chen PII: DOI: Reference:
S0168-1605(15)00237-8 doi: 10.1016/j.ijfoodmicro.2015.04.034 FOOD 6899
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
17 November 2014 17 March 2015 19 April 2015
Please cite this article as: Ma, Chengjie, Ma, Aimin, Gong, Guangyu, Liu, Zhenmin, Wu, Zhengjun, Guo, Benheng, Chen, Zhengjun, Cracking Streptococcus thermophilus to stimulate the growth of the probiotic Lactobacillus casei in co-culture, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.04.034
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Cracking Streptococcus thermophilus to stimulate the growth of the probiotic
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Lactobacillus casei in co-culture
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Chengjie Maa, b, Aimin Mac, Guangyu Gonga, Zhenmin Liua, Zhengjun Wua, Benheng Guoa, * , Zhengjun Chend, *
State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy &
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a
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Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai 200436, China Wuhan Bright Dairy Co., Ltd., 1 Zhangbai Road, Wuhan 430040, China
c
College of Food Science and Technology, Huazhong Agricultural University, No.1
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b
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Shizishan Street, Wuhan 430070, China
State Key Laboratory of Agricultural Microbiology, College of Life Science and
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Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan 430070,
*
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China
Corresponding author
Zhengjun Chen
College of Life Science and Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan 430070, China E-mail: [email protected]; Tel.: 86-27-87281267; Fax: 86-27-87280670 Benheng Guo State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai 200436, China E-mail: [email protected]; Tel.: 86-21-66308987; Fax: 86-21-66308968 1
ACCEPTED MANUSCRIPT ABSTRACT Lactobacillus casei, a probiotic, and Streptococcus thermophilus, a fast acidifying lactic
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acid bacterial strain, are both used in the food industry. The aim of this study was to
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investigate the interaction between L. casei and S. thermophilus in the presence or absence of S. thermophilus-specific bacteriophage during milk fermentation. The acidification
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capability of L. casei co-cultured with S. thermophilus was significantly higher than that observed for L. casei or S. thermophilus cultured alone. However, the probiotic content
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(i.e., L. casei cell viability) was low. The fastest acidification and the highest viable L. casei cell count were observed in co-cultures of L. casei and S. thermophilus with S.
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thermophilus phage. In these co-cultures, S. thermophilus compensated for the slow acid
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production of L. casei in the early exponential growth phase. Thereafter, phage-induced
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lysis of the S. thermophilus cells eliminated the competition for nutrients, allowing L. casei to grow well. Additionally, the ruptured S. thermophilus cells released intracellular
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factors, which further promoted the growth and function of the probiotic bacteria. Crude cellular extract isolated from S. thermophilus also significantly accelerated the growth and propagation of L. casei, supporting the stimulatory role of the phage on this micro-ecosystem.
Keywords: Lactobacillus casei; Streptococcus thermophilus; Streptococcus thermophilus phage; co-culture; fermented milk
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1. Introduction
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Lactobacillus casei is traditionally recognized as a probiotic, and has been
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substantially utilized in the food industry (Sharma and Devi, 2014). The health-promoting and nutritional properties of L. casei have been investigated in several published clinical
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studies, which support the beneficial role of these bacteria in multiple biological processes, including regulation of the immune system, inhibition of intestinal pathogens, and
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prevention of cardiovascular disease (Awaisheh et al., 2013; Liévin-Le Moal and Servin, 2014; Wang et al., 2013). These demonstrated health benefits have led to prevailing
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consumer demand for this probiotic. In general, milk is considered to be a good carrier of probiotics, and large-scale fermentation of L. casei has been used to manufacture
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probiotic-fermented dairy products. However, it has been shown that the reported L. casei strains exhibit low growth activity in milk (Ma et al., 2015; Zalán et al., 2010). The
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commercially available fermented dairy products that use L. casei cultures, such as ActimelTM, are very time-consuming to produce (Zaręba et al., 2014). Therefore, it is necessary to resolve the growth of probiotic L. casei in milk and enhance the production of probiotic-containing dairy products for consumers. Mixed-strain culture fermentation is an effective approach to obtain the desired product characteristics and to reduce fermentation time in most food fermentation processes (Laiño et al., 2014; Smid and Lacroix, 2013). For example, yogurt is typically produced using mixed starter cultures comprised of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. This simple, but successful, ecosystem relies 3
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on the metabolite exchange of purines, amino acids, and long-chain fatty acids (Sieuwerts
bacteria.
Lactobacillus
rhamnosus,
Lactobacillus
acidophilus,
and
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probiotic
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et al., 2008). However, this system does not enhance the growth and reproduction of all
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Bifidobacterium lactis appear to be inhibited when co-cultured with the fast-acidifying strain S. thermophilus, resulting in a low probiotic content of the final product (Oliveira et
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al., 2009). Vinderola et al. (2002) assessed the interactions among lactic acid bacteria (LAB) and probiotic bacteria and found that the cell-free supernatant (CFS) obtained from
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skim milk cultures fermented by S. thermophilus and L. bulgaricus had no positive effect on probiotic L. casei, indicating that the extracellular products secreted by these yogurt
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bacteria were not conducive to the growth of L. casei. However, the crude cellular extract
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(CCE) obtained from the L. bulgaricus cultures, containing β-galactosidase and protease
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inherent to the bacteria, did appear to stimulate the growth of the probiotic L. rhamnosus in milk (Gaudreau et al., 2005). This study provided valuable insight into the use of
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―cracked‖ yogurt bacteria cells to accelerate the growth of probiotic bacteria. It is well known that the yogurt bacteria S. thermophilus and L. bulgaricus are susceptible to their corresponding virulent bacteriophages during the milk fermentation process. Thus, phage predation is an effective way to lyse these yogurt bacteria cells, which hinted that co-culturing a probiotic with S. thermophilus and rupturing the S. thermophilus cells using phage could potentially promote the fermentation and stimulate the growth of probiotic cultures. However, there is very little information concerning the effect of S. thermophilus on the growth of L. casei in co-cultures of these two species in milk, especially in the presence of S. thermophilus phage. 4
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In this study, the interactions between L. casei and S. thermophilus in the presence or
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absence of S. thermophilus bacteriophage during milk fermentation were investigated. The
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influence of the S. thermophilus cell lysates on the growth and propagation of the L. casei
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cultures was evaluated. Fast acidification and considerable cell density of L.casei were achieved. To our knowledge, this is the first report of L. casei co-cultured with S.
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thermophilus in the presence of S. thermophilus phage.
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2. Materials and methods
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2.1. Bacterial strains and culture conditions
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L. casei LC2W, a probiotic strain that exhibits antihypertensive effects (Ai et al., 2008), was isolated from a traditional dairy product in Inner Mongolia, China. The L. casei cultures were grown using MRS (Oxoid, Basingstoke, England) under anaerobic
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conditions in 2.5 L anaerobic jars using AnaeroGENTM sachets (Oxoid, Basingstoke, England) at 37°C. S. thermophilus ST1 was isolated from the commercial Direct Vat Set yogurt starter, and phage φ101 was identified as its virulent bacteriophage (Ma et al., 2014). S. thermophilus was grown using M17 (Oxoid, Basingstoke, England) supplemented with 0.5% (w/v) lactose (LM17) in an MIR-253 incubator (Sanyo, Osaka, Japan) at 42°C. The L. casei and S. thermophilus strains were prepared as direct vat-set cultures (2 × 1011 colony-forming units (CFU)/g) in our laboratory. Preparation of the phage lysate and phage counting were carried out according to Quiberoni et al. (2006).
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2.2. L. casei co-culturing with S. thermophilus in the presence or absence of S.
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thermophilus phage
Skim milk powder (33.4% protein, 0.8% fat; Fonterra Ltd., Auckland, New Zealand)
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was reconstituted in distilled water (reconstituted skim milk, RSM). The RSM (12%, w/w) was then heated to 95°C for 90 min using a GFL1002 water-bath (GFL Company,
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Burgwedel, Germany). After being cooled to 37°C, the prepared RSM was inoculated with L. casei LC2W (5 × 106 CFU/g) and S. thermophilus ST1 (5 × 106 CFU/g). Cultures
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without phage were designated LC-ST, while those supplemented with S. thermophilus
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phage φ101 were designated LC-ST-P. RSM was inoculated with single L. casei LC2W (5
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× 106 CFU/g or 1 × 107 CFU/g) or S. thermophilus ST1 (5 × 106 CFU/g or 1 × 107 CFU/g) as a control (designated LC and ST). The samples of RSM fermented by L. casei LC2W
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(designated LC-P) or S. thermophilus ST1 (designated ST-P) in the presence of S. thermophilus phage were also cultured and analyzed. All of the fermented milk cultures were incubated at 37°C and were monitored automatically (recorded every 5 min) for pH using a Cinac system (Alliance Instruments, Mery-Sur-Oise, France) during milk acidification. The number of viable L. casei and S. thermophilus cells were selectively counted during fermentation using MRS (fermented milk sampled every 12 h for 72 h) and LM17 (fermented milk sampled every 2 h for 24 h) agar (Ashraf and Shah, 2011), respectively.
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2.3. Preparation of S. thermophilus CFS
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S. thermophilus ST1 was grown in 12% RSM at 37°C for 24 h. The fermented milk
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was centrifuged at 8000 × g for 10 min at 5°C. The supernatant was adjusted to a pH of 6.5 using 1 M NaOH and then filtered through a 0.22-μm pore-size filter (Millipore,
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Billerica, MA, USA) for sterilization. The CFS was lyophilized and kept frozen (-80°C)
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until use.
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2.4. S. thermophilus cell collection and production of CCE
S. thermophilus cell collection was carried out according to Gaudreau et al. (2005),
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and production of the CCE was carried out according to Tanigawa et al. (2010) with some modifications. A total of 10 L of sterile (110°C for 15 min) RSM (12%, w/w) was
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inoculated with S. thermophilus ST1 (1 × 107 CFU/g) and cultured at 37°C in a BLBIO-10SJ fermenter (Bailun, Shanghai, China). Agitation of the cultures was kept at 50 rpm, and the pH (maintained at 5.6) was buffered with 5 N ammonium hydroxide. Fermentations were stopped after 24 h of incubation. Hereafter, the viable S. thermophilus cells were counted using LM17 agar. Cell pellets were collected by centrifugation (8000 × g for 10 min at 5°C) and washed thrice in TMA-I buffer (10 mM Tris-HCl, pH 7.8, 30 mM NH4Cl, 10 mM MgCl2, and 6 mM 2-mercaptoethanol) in order to avoid the precipitation of casein during centrifugation. After washing twice in sterile saline (0.85%, w/w), the cell pellets were ground on four separate, consecutive occasions: beating for 20 s each at 3000 7
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rpm with 0.1-mm zirconia silica beads in a Biospec mini bead-beater-16 (Bartlesville, OK, USA), after removing the beads and cell debris by centrifugation, the cell lysates were
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adjusted to 5 × 109 ruptured S. thermophilus cells/g using distilled water according to the
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aforementioned S. thermophilus viable cell count. Then, lysates were filtered through a 0.22-μm pore-size filter for sterilization. The obtained CCE was kept frozen (-80°C) ready
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for use.
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2.5. Effect of S. thermophilus CFS and CCE on L. casei
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The growth-promoting properties of CFS and CCE isolated from S. thermophilus on L. casei LC2W were determined by evaluating the acidification performance and L. casei
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cell viability. Different concentrations of heated (sterilized at 110°C for 15 min) or native (sterilized using a filter) CFS and CCE were added to sterile RSM (final concentration of
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RSM, 12%, w/w) to prepare the media. The CFS- and CCE-supplemented media were then inoculated with L. casei LC2W (1 × 107 CFU/g) and incubated at 37°C. RSM without supplementation was inoculated with L. casei LC2W as a control. The pH of each culture was measured with a 420A pH-meter (Thermo Orion, Beverly, MA, USA) and the viable cells were counted using MRS agar every 24 h.
2.6. Statistical analysis
All physicochemical experiments were performed in triplicate, and the results are 8
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expressed as the mean or means ± standard deviation (SD). Significant differences among
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treatments were evaluated by performing analysis of variance (ANOVA) F tests and
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Fisher's least significant difference (p < 0.05) using Statgraphics Plus 5.1 software
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(Manugistics, Rockville, MD, USA).
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3. Results and discussion
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3.1. Acidification performance of L. casei co-cultured with S. thermophilus
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L. casei and S. thermophilus presented different acidification rates in milk(Fig. 1).
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The pH of the fermented milk rapidly decreased after inoculation with a single strain of S.
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thermophilus (ST). However, the acidification slowed down when the pH dropped to around 5.0, and completely halted at pH 4.4. The pH of the fermented milk inoculated
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with L. casei alone (LC) decreased slowly in the early fermentation stage, but fast acidification was observed after incubating for 6 h. Co-culture of L. casei and S. thermophilus (LC-ST) appeared to combine the advantages of the two strains. In these co-cultures, the pH of the inoculated fermented milk decreased constantly throughout the incubation, indicating that the dual inoculators had a higher level of acidification capability. In general, LAB such as S. thermophilus and L. bulgaricus possess strong β-galactosidase and protease activities and they are fast-acting acidifiers in milk (Shah and Jelen, 1991). Unfortunately, probiotic bacteria such as L. casei usually possess weak
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β-galactosidase,
phospho-β-galactosidase,
protease
activities
and
lactose
phospho-transferase system (Ikram ul and Mukhtar, 2006; Mozzi et al., 2009; Wu et al.,
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2009). Notably, phospho- and β-galactosidase, as well as the entire proteolytic system, are
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crucial for LAB and probiotic bacteria to grow in milk. These enzymes are usually located in the cell wall or intracellular compartment (Savijoki et al., 2006). Mixture of selected
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bacterial strains could be adopted to obtain desirable characteristics such as high probiotic viable cells, fast acidification, and good flavor. For the intrinsic intracellular proteolytic
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enzymes, it is possibly beneficial to lyse the cells to exploit them when co-cultured with probiotic bacteria. Therefore, a specific bacteriophage for S. thermophilus was
L. casei (LC-ST-P).
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supplemented to the co-cultures to investigate the interaction between S. thermophilus and
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The acidity produced by these bacteriophage-spiked co-cultures was slightly lower than that observed from the LC-ST co-cultures without phage during the first 24 h, which
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might be due to phage invasion that weakened the acidification ability of the S. thermophilus strain. Virulent S. thermophilus phage φ101was previously shown to be destructive to a corresponding sensitive strain during fermentation (Ma et al., 2014). In the present study, the pH of S. thermophilus with phage (ST-P) decreased during the first 9 h after inoculation. However, the acidification completely halted at pH 5.3 because of the reproduction and releasing of bacteriophages (Fig. 1). Interestingly, the decrease in pH of the LC-ST-P cultures surpassed that of the LC-ST cultures from 24 to 72 h, indicating an enhanced level of acidification. For this reason, we thought that phage invasion introduced the intracellular lysate of S. thermophilus into the media, which stimulated the growth and 10
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acidification ability of L. casei. The acidification curves of LC were mostly coincident
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with that of LC-P (samples fermented by L. casei in the presence of S. thermophilus phage,
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acidification curves not shown), which demonstrated that the phages of S. thermophilus
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had no effect on L. casei. Overall, the co-cultures especially in the presence of phage shortened the fermentation time. For example, the pH value of LC fermented milk was
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3.72 at 72 h. However, the fermentation times reaching the same pH end point (3.72) in LC-ST and LC-ST-P fermented milk were only 61 h and 40 h, respectively (Fig. 1). The
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acidification performance was promoted by L. casei co-cultured with phage-lysed S. thermophilus, which provided a feasible and effective method to improve probiotic
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fermentation.
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3.2. Growth and propagation of L. casei co-cultured with S. thermophilus
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The numbers of L. casei found in the LC, LC-ST, and LC-ST-P fermented milk cultures at 72 h were 8.83, 7.31, and 9.35 log CFU/g, respectively (Fig. 2). Compared to the LC fermented milk samples, a significant decrease of 1.52 log CFU/g was observed in the LC-ST fermented milk samples, indicating that L. casei cell viability was affected by co-cultured S. thermophilus during milk fermentation. It seemed that the probiotic L. casei was inhibited by the metabolites secreted by S. thermophilus (for example, organic acids and antimicrobial agents). Another possible explanation of this bacterial population changes was that the added S. thermophilus was competitive for the nutrients present in the milk. When the phage was added to the co-cultivation, the number of viable L. casei at 11
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72 h increased by 0.52 and 2.04 log CFU/g compared to the LC and LC-ST cultures,
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respectively (Fig. 2). It was consistent with the assumption that the decrease in the number
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of viable S. thermophilus bacteria caused by the phage invasion eliminated the competitive
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inhibition of L. casei in late fermentation, resulting in an increase in the viability of L. casei in the co-culture. Indeed, compared to LC, the slight increase in L. casei cell count in
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the LC-ST-P cultures indicated that lysing and releasing of the cellular contents of S. thermophilus by the virulent phage stimulated the growth and propagation of L. casei.
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These results were also consistent with the changes observed for the acidification performance, whereby the phage-supplemented co-cultures showed the strongest
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acidification ability.
S. thermophilus is a LAB that has the ability to quickly acidify milk. In fermented
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milk samples containing only S. thermophilus (ST), the bacteria entered the exponential growth phase after incubating for 2 h and the cells number of S. thermophilus peaked after
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12 h (Fig. 3). In the co-cultured experiments (LC-ST), the change in the number of S. thermophilus mirrored the changes observed when S. thermophilus was cultured alone. In the LC-ST-P samples, S. thermophilus grew and reproduced rapidly in the early stage of fermentation (2–8 h), as expected, and decreased very quickly in the later stages of fermentation following phage invasion (Fig. 3). The S. thermophilus cell count of ST-P fermented milk increased rapidly in the early stage of fermentation and decreased quickly in the later stages of fermentation (data not shown), which was analogous to the change observed in the ST-LC-P. Taken together, these results suggest that while the acidification performance of the 12
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co-culture LC-ST was significantly higher than that of the single LC or ST cultures, the
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probiotic content of L. casei was rather low, which indicated that simple co-cultivation
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was impractical to enhance the probiotic production in customer dairy products.
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Fortunately, the fastest acidification and the highest L. casei cell count were observed in the phage-supplemented co-culture experiments. This provided a straightforward approach
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to increase the probiotic content of this dairy product while simultaneously shortening the fermentation time. Furthermore, other probiotic strains such as Lactobacillus plantarum, L.
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rhamnosus, and Bifidobacterium sp. also showed stimulated growth and propagation when co-cultured with S. thermophilus spiked with specific phage (data not shown), which
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suggested that this co-culturing technique could be applied widely to promote various probiotic bacteria fermentation. Detailed investigation was needed for the large-scale
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application of this system in the food industry. Furthermore, discovering the mechanism of this enhanced L. casei probiotic content and acidification by the lysed S. thermophilus is
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essential to the implementation of this technique.
3.3. Acidification performance of L. casei supplemented with CFS and CCE
In order to elucidate the role of extracellular and intracellular metabolites and signaling molecules, the effects of the CFS and CCE isolated from S. thermophilus on the cultured L. casei bacteria were investigated. The CFS was known to contain the metabolites and secretory components utilized by S. thermophilus during milk fermentation. Therefore, determining the effects of isolated CFS on L. casei could reveal 13
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the interactions between L. casei and the extracellular metabolites of S. thermophilus. The
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CCE contains the lysed intracellular products of S. thermophilus cells. Determining the
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effect of CCE on L. casei could demonstrate the interactions occurring between L. casei
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and the intracellular metabolites of S. thermophilus in the presence of S. thermophilus phage. Thus, cracked and un-cracked, CCE and CFS of S. thermophilus were added into
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RSM during L. casei fermentation, and the acidification and propagation of L. casei were evaluated.
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As shown in Table 1, the addition of native CCE, at a final concentration of 0.2–2.0% (w/w), to the RSM-L. casei culture induced a statistically significant change in pH (p
0.05) were observed only adding 0.02% native CCE. Furthermore, heat greatly diminished the effect of CCE on the acidification performance in the L. casei cultures (for example, CCEN2.0% decreased pH by 2.10 vs. only 1.58 for CCEH2.0% at 24 h). These data suggested that a large proportion of the intracellular components isolated from S. thermophilus were heat-inactivated components such as biological active enzymes, while a smaller proportion were likely composed of non-temperature-dependent chemical molecules such as growth factors. In regards to the role of extracellular S. thermophilus molecules, no statistically significant differences (p > 0.05) in the acidification performance were observed following 14
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the addition of either native or heated CFS, at a final concentration of 0.02–2.0% (w/w) in
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12% RSM compared to the untreated control cultures (data not shown). These results are
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consistent with Vinderola et al. (2002), who also found that the CFS obtained from skim
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milk cultures fermented by S. thermophilus had no positive effect on probiotic L. casei.
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3.4. Growth and propagation of L. casei supplemented with CFS and CCE
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In addition to investigating the effect of CFS and CCE on the L. casei-induced acidification, we also studied the growth response of this bacterium to the CFS and CCE at
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different concentrations. 0.2–2.0% native CCE and 2.0% heated CCE isolated from S. thermophilus clearly enhanced the growth and propagation of L. casei in these
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experiments. The viable L. casei counts of CCEH2.0%, CCEN0.2%, and CCEN2.0% were 9.16, 9.28, and 9.40 log CFU/g, increase in cell viability at 0.33, 0.45, and 0.57 log CFU/g,
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compared to the control at 72 h (Table 1), respectively. These results are similar to those found for the phage-lysed S. thermophilus, whereby the growth and propagation of the co-cultured L. casei were enhanced. We suspect that the same components and signaling mechanism caused this change in viability after the addition of CCE. However, the addition of 0.02% native CCE (i.e., 106 ruptured S. thermophilus cells/g) showed no significant differences (p > 0.05). Therefore, the stimulatory growth and propagation on L. casei could be observed only when enough CCE was added
(more than 107 ruptured S.
thermophilus cells/g). Similar to the results from the acidification experiments, no statistically significant 15
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differences (p > 0.05) in cell viability were observed following the addition of native or
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heated CFS (data not shown). CFS is known to contain low levels of free amino acids and
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soluble peptides, which benefits for cell growth and viability (Serra et al., 2009; Vinderola
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et al., 2002). Zhang et al. (2010) showed that milk peptides could promote the growth of LAB. However, it is possible that the peptide content in the CFS isolated in this study was
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too low to stimulate L. casei effectively. Additional work is necessary to fully determine the role of the extracellular components of S. thermophilus CFS in L. casei.
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In brief, we showed that S. thermophilus could be used to compensate for the slow acid-producing ability of L. casei. S. thermophilus-specific bacteriophage, used to rupture
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viable S. thermophilus cells, was also shown to stimulate the growth and propagation of L. casei. Notably, a reduced fermentation time and high L. casei cell count were achieved
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during fermentation in the co-cultures. Although phages are considered to be unfavorable environmental factors and retard fermentation in yogurt and cheese manufacturing (Zinno
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et al., 2010), they played a unique and positive role in lysing their corresponding LAB and releasing the intracellular products during the production of fermented milk with high probiotic content. We believe that the addition of S. thermophilus to the probiotic-enriched milk can enhance the consumer product two-fold. First, S. thermophilus is a fast-acidifying strain in milk and compensates for the slow acid-producing ability of L. casei, especially in the early exponential growth phase, which helps to rapidly reduce the pH of the fermented milk and avoid the risk of undesirable microorganism growth. Second, L. casei is a fastidious microorganism (Cai et al., 2009; Du Toit et al., 2011) and the pool of free amino acids and peptides in milk is not sufficient to fulfill optimal bacterial growth. Thus, 16
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the phage-induced release of the S. thermophilus cell lysate, which contains proteases,
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lactase, free amino acids, and peptides, accelerates the growth and propagation of the
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probiotic. Probiotic growth is then further enhanced, as the decrease in viable S.
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thermophilus cells also eliminates the competition between this bacterium and the probiotic for nutrients in the media.
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This study is the first to investigate the relationship between co-cultured S. thermophilus and L. casei as well as the effects of S. thermophilus-derived CFS and CCE
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on the cell count and milk fermentation capabilities of L. casei. Research concerning probiotics, LAB/probiotic co-culture techniques, and dairy product fermentation is far
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from complete, and additional work to uncover the full relationship between LAB and
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probiotic bacteria is warranted.
Acknowledgements
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This work was supported by National High Technology Research and Development Program of China (2011AA100901), and the National Key Technologies Program of China (2013BAD18B01) during the 12th Five-Year Plan Period.
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kinetic analysis of fermentation parameters. Indian Journal of Biochemistry &
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Laiño, J.E., Juarez del Valle, M., Savoy de Giori, G., LeBlanc, J.G., 2014. Applicability of a Lactobacillus amylovorus strain as co-culture for natural folate bio-enrichment of
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fermented milk. International Journal of Food Microbiology 191, 10-16. Liévin-Le Moal, V., Servin, A.L., 2014. Anti-infective activities of Lactobacillus strains in
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the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clinical Microbiology Reviews 27, 167-199.
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Ma, C., Gong, G., Liu, Z., Ma, A., Chen, Z., 2015. Stimulatory effects of tea supplements on the propagation of Lactobacillus casei in milk. International Dairy Journal 43, 1-6.
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Ma, C., Pan, N., Chen, Z., Liu, Z., Gong, G., Ma, A., 2014. Geographical diversity of Streptococcus thermophilus phages in Chinese yoghurt plants. International Dairy
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Journal 35, 32-37.
Mozzi, F., Gerbino, E., Font de Valdez, G., Torino, M.I., 2009. Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system. Journal of Applied Microbiology 107, 56-64. Oliveira, R.P.S., Perego, P., Converti, A., Oliveira, M.N., 2009. Growth and acidification performance of probiotics in pure culture and co-culture with Streptococcus thermophilus: the effect of inulin. LWT-Food Science Technology 42, 1015-1021.
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Diversity of Streptococcus thermophilus phages in a large-production cheese factory
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in Argentina. Journal of Dairy Science 89, 3791-3799.
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Savijoki, K., Ingmer, H., Varmanen, P., 2006. Proteolytic systems of lactic acid bacteria. Applied Microbiology and Biotechnology 71, 394-406.
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Serra, M., Trujillo, A.J., Guamis, B., Ferragut, V., 2009. Proteolysis of yogurts made from ultra-high-pressure homogenized milk during cold storage. Journal of Dairy Science
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92, 71-78.
Shah, N.P., Jelen, P., 1991. Lactase activity and properties of sonicated dairy cultures.
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Milchwissenschaft-Milk Science International 46, 570-573. Sharma, M., Devi, M., 2014. Probiotics: a comprehensive approach toward health foods.
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Critical Reviews in Food Science and Nutrition 54, 537-552. Sieuwerts, S, de Bok, F.A., Hugenholtz, J., van Hylckama Vlieg, J.E., 2008. Unraveling
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microbial interactions in food fermentations: from classical to genomics approaches. Applied and Environmental Microbiology 74, 4997-5007. Smid, E.J., Lacroix, C., 2013. Microbe-microbe interactions in mixed culture food fermentations. Current Opinion in Biotechnology 24, 148-154. Tanigawa, K., Kawabata, H., Watanabe, K., 2010. Identification and typing of Lactococcus lactis by matrix-assisted laser desorption ionization–time of flight mass spectrometry. Applied and Environmental Microbiology 76, 4055-4062.
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Vinderola, C.G., Mocchiutti, P., Reinheimer, J.A., 2002. Interactions among lactic acid
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starter and probiotic bacteria used for fermented dairy products. Journal of Dairy
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Science 85, 721-729.
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Wang, Y., Xie, J., Wang, N., Li, Y., Sun, X., Zhang, Y., Zhang, H., 2013. Lactobacillus casei Zhang modulate cytokine and toll-like receptor expression and beneficially
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regulate poly I:C-induced immune responses in RAW264.7 macrophages. Microbiology and Immunology 57, 54-62.
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Wu, R., Wang, W., Yu, D., Zhang, W., Li, Y., Sun, Z., Wu, J., Meng, H., Zhang, H., 2009. Proteomics analysis of Lactobacillus casei Zhang, a new probiotic bacterium isolated
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from traditional home-made koumiss in Inner Mongolia of China. Molecular & Cellular Proteomics MCP8, 2321-2338.
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Zalán, Z., Hudáček, J., Štětina, J., Chumchalová, J., Halász, A., 2010. Production of organic acids by Lactobacillus strains in three different media. European Food
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Research and Technology 230, 395-404. Zaręba, D., Ziarno, M., Scibisz, I., Gawron, J., 2014. The importance of volatile compound profile in the assessment of fermentation conducted by Lactobacillus casei DN-114001. International Dairy Journal 35, 11-14. Zhang, Q., Zhao, M., Qu, D., Zhao, H., Zhao, Q., 2010. Effect of papain-hydrolysed casein peptides on the fermentation kinetics, microbiological survival and physicochemical properties of yoghurt. International Journal of Food Science and Technology 45, 2379-2386. Zinno, P., Janzen, T., Bennedsen, M., Ercolini, D., Mauriello, G., 2010. Characterization of 21
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Streptococcus thermophilus lytic bacteriophages from mozzarella cheese plants.
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International Journal of Food Microbiology 138, 137-144.
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Table:
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Table 1 The influence of CCE on pH values and viable L. casei counts of fermented milk
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Figure legends:
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Fig. 1 Acidification curves during fermentation using different starter cultures ST: Fermented by S. thermophilus; LC: Fermented by L. casei; LC-ST: L. casei
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co-culturing with S. thermophilus; LC-ST-P: L. casei co-culturing with S. thermophilus in the presence of S. thermophilus phages; ST-P: Fermented by S. thermophilus with S.
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thermophilus phages.
All the samples fermented by L. casei/S. thermophilus at total inoculum of 1×107 CFU/g
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The phage inoculum size was 1×102 PFU/g.
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Fig. 2 Changes in viable L. casei counts during milk fermentation LC: inoculated with single L. casei (5 × 106 CFU/g); LC-ST: inoculated with L. casei (5 ×
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106 CFU/g) and S. thermophilus (5 × 106 CFU/g); LC-ST-P: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g) in the presence of S. thermophilus phages (1×102 PFU/g).
Fig. 3 Changes in viable S. thermophilus counts during milk fermentation ST: inoculated with single S. thermophilus (5 × 106 CFU/g); LC-ST: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g); LC-ST-P: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g) in the presence of S. thermophilus phages (1×102 PFU/g). 23
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Figure 1
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Figure 2
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Figure 3
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pH values
L. casei counts (log CFU/g)
Sampls 24 h
48 h
72 h
RSM
6.36±0.03a
4.98±0.06 a
3.86±0.05 a
3.74±0.03 a
8.83±0.16 b
CCEN0.02%
6.36±0.03 a
4.92±0.05 a
3.81±0.04 a
3.73±0.03 a
8.89±0.15 b
CCEN0.2%
6.36±0.05 a
4.61±0.06 c
3.69±0.06 b
3.59±0.02 b
9.28±0.10 a
CCEN2.0%
6.38±0.03 a
4.28±0.04 d
3.65±0.04 b
3.57±0.03 b
9.40±0.17 a
CCEH0.02%
6.35±0.03 a
4.99±0.05 a
3.87±0.07 a
3.75±0.03 a
8.82±0.12 b
CCEH0.2%
6.37±0.05 a
4.97±0.07 a
3.86±0.06 a
3.73±0.04 a
8.87±0.11 b
CCEH2.0%
6.38±0.04 a
4.80±0.05 b
3.71±0.05 b
3.62±0.03 b
9.16±0.14 a
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0h
RSM: fermented milk without supplementation (control); CCEN0.02%, CCEN0.2% and CCEN2.0%:
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fermented milk containing 0.02%, 0.2% and 2.0% native CCE; CCEH0.02%, CCEH0.2% and CCEH2.0%:
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fermented milk containing 0.02%, 0.2% and 2.0% heated CCE; The L. casei counts are the values measured at 72 h. All values are mean ± SD (n=3). Rows with different superscript letters indicate
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statistical differences (P < 0.05).
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Highlights
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1. S. thermophilus compensated for the slow acid production of L. casei.
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2. Phage-induced lysis eliminated the competitive inhibition on L. casei.
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3. The lysate of S. thermophilus stimulated the growth and propagation of L. casei.
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4. The effect of CCE on L. casei confirmed the stimulatory role of the phage.
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S0168-1605(15)00237-8 doi: 10.1016/j.ijfoodmicro.2015.04.034 FOOD 6899
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
17 November 2014 17 March 2015 19 April 2015
Please cite this article as: Ma, Chengjie, Ma, Aimin, Gong, Guangyu, Liu, Zhenmin, Wu, Zhengjun, Guo, Benheng, Chen, Zhengjun, Cracking Streptococcus thermophilus to stimulate the growth of the probiotic Lactobacillus casei in co-culture, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.04.034
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Cracking Streptococcus thermophilus to stimulate the growth of the probiotic
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Lactobacillus casei in co-culture
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Chengjie Maa, b, Aimin Mac, Guangyu Gonga, Zhenmin Liua, Zhengjun Wua, Benheng Guoa, * , Zhengjun Chend, *
State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy &
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a
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Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai 200436, China Wuhan Bright Dairy Co., Ltd., 1 Zhangbai Road, Wuhan 430040, China
c
College of Food Science and Technology, Huazhong Agricultural University, No.1
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b
d
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Shizishan Street, Wuhan 430070, China
State Key Laboratory of Agricultural Microbiology, College of Life Science and
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Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan 430070,
*
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China
Corresponding author
Zhengjun Chen
College of Life Science and Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan 430070, China E-mail: [email protected]; Tel.: 86-27-87281267; Fax: 86-27-87280670 Benheng Guo State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd., 1518 Jiangchang Road (W), Shanghai 200436, China E-mail: [email protected]; Tel.: 86-21-66308987; Fax: 86-21-66308968 1
ACCEPTED MANUSCRIPT ABSTRACT Lactobacillus casei, a probiotic, and Streptococcus thermophilus, a fast acidifying lactic
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acid bacterial strain, are both used in the food industry. The aim of this study was to
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investigate the interaction between L. casei and S. thermophilus in the presence or absence of S. thermophilus-specific bacteriophage during milk fermentation. The acidification
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capability of L. casei co-cultured with S. thermophilus was significantly higher than that observed for L. casei or S. thermophilus cultured alone. However, the probiotic content
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(i.e., L. casei cell viability) was low. The fastest acidification and the highest viable L. casei cell count were observed in co-cultures of L. casei and S. thermophilus with S.
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thermophilus phage. In these co-cultures, S. thermophilus compensated for the slow acid
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production of L. casei in the early exponential growth phase. Thereafter, phage-induced
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lysis of the S. thermophilus cells eliminated the competition for nutrients, allowing L. casei to grow well. Additionally, the ruptured S. thermophilus cells released intracellular
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factors, which further promoted the growth and function of the probiotic bacteria. Crude cellular extract isolated from S. thermophilus also significantly accelerated the growth and propagation of L. casei, supporting the stimulatory role of the phage on this micro-ecosystem.
Keywords: Lactobacillus casei; Streptococcus thermophilus; Streptococcus thermophilus phage; co-culture; fermented milk
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1. Introduction
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Lactobacillus casei is traditionally recognized as a probiotic, and has been
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substantially utilized in the food industry (Sharma and Devi, 2014). The health-promoting and nutritional properties of L. casei have been investigated in several published clinical
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studies, which support the beneficial role of these bacteria in multiple biological processes, including regulation of the immune system, inhibition of intestinal pathogens, and
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prevention of cardiovascular disease (Awaisheh et al., 2013; Liévin-Le Moal and Servin, 2014; Wang et al., 2013). These demonstrated health benefits have led to prevailing
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consumer demand for this probiotic. In general, milk is considered to be a good carrier of probiotics, and large-scale fermentation of L. casei has been used to manufacture
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probiotic-fermented dairy products. However, it has been shown that the reported L. casei strains exhibit low growth activity in milk (Ma et al., 2015; Zalán et al., 2010). The
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commercially available fermented dairy products that use L. casei cultures, such as ActimelTM, are very time-consuming to produce (Zaręba et al., 2014). Therefore, it is necessary to resolve the growth of probiotic L. casei in milk and enhance the production of probiotic-containing dairy products for consumers. Mixed-strain culture fermentation is an effective approach to obtain the desired product characteristics and to reduce fermentation time in most food fermentation processes (Laiño et al., 2014; Smid and Lacroix, 2013). For example, yogurt is typically produced using mixed starter cultures comprised of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. This simple, but successful, ecosystem relies 3
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on the metabolite exchange of purines, amino acids, and long-chain fatty acids (Sieuwerts
bacteria.
Lactobacillus
rhamnosus,
Lactobacillus
acidophilus,
and
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probiotic
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et al., 2008). However, this system does not enhance the growth and reproduction of all
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Bifidobacterium lactis appear to be inhibited when co-cultured with the fast-acidifying strain S. thermophilus, resulting in a low probiotic content of the final product (Oliveira et
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al., 2009). Vinderola et al. (2002) assessed the interactions among lactic acid bacteria (LAB) and probiotic bacteria and found that the cell-free supernatant (CFS) obtained from
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skim milk cultures fermented by S. thermophilus and L. bulgaricus had no positive effect on probiotic L. casei, indicating that the extracellular products secreted by these yogurt
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bacteria were not conducive to the growth of L. casei. However, the crude cellular extract
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(CCE) obtained from the L. bulgaricus cultures, containing β-galactosidase and protease
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inherent to the bacteria, did appear to stimulate the growth of the probiotic L. rhamnosus in milk (Gaudreau et al., 2005). This study provided valuable insight into the use of
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―cracked‖ yogurt bacteria cells to accelerate the growth of probiotic bacteria. It is well known that the yogurt bacteria S. thermophilus and L. bulgaricus are susceptible to their corresponding virulent bacteriophages during the milk fermentation process. Thus, phage predation is an effective way to lyse these yogurt bacteria cells, which hinted that co-culturing a probiotic with S. thermophilus and rupturing the S. thermophilus cells using phage could potentially promote the fermentation and stimulate the growth of probiotic cultures. However, there is very little information concerning the effect of S. thermophilus on the growth of L. casei in co-cultures of these two species in milk, especially in the presence of S. thermophilus phage. 4
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In this study, the interactions between L. casei and S. thermophilus in the presence or
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absence of S. thermophilus bacteriophage during milk fermentation were investigated. The
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influence of the S. thermophilus cell lysates on the growth and propagation of the L. casei
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cultures was evaluated. Fast acidification and considerable cell density of L.casei were achieved. To our knowledge, this is the first report of L. casei co-cultured with S.
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thermophilus in the presence of S. thermophilus phage.
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2. Materials and methods
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2.1. Bacterial strains and culture conditions
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L. casei LC2W, a probiotic strain that exhibits antihypertensive effects (Ai et al., 2008), was isolated from a traditional dairy product in Inner Mongolia, China. The L. casei cultures were grown using MRS (Oxoid, Basingstoke, England) under anaerobic
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conditions in 2.5 L anaerobic jars using AnaeroGENTM sachets (Oxoid, Basingstoke, England) at 37°C. S. thermophilus ST1 was isolated from the commercial Direct Vat Set yogurt starter, and phage φ101 was identified as its virulent bacteriophage (Ma et al., 2014). S. thermophilus was grown using M17 (Oxoid, Basingstoke, England) supplemented with 0.5% (w/v) lactose (LM17) in an MIR-253 incubator (Sanyo, Osaka, Japan) at 42°C. The L. casei and S. thermophilus strains were prepared as direct vat-set cultures (2 × 1011 colony-forming units (CFU)/g) in our laboratory. Preparation of the phage lysate and phage counting were carried out according to Quiberoni et al. (2006).
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2.2. L. casei co-culturing with S. thermophilus in the presence or absence of S.
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thermophilus phage
Skim milk powder (33.4% protein, 0.8% fat; Fonterra Ltd., Auckland, New Zealand)
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was reconstituted in distilled water (reconstituted skim milk, RSM). The RSM (12%, w/w) was then heated to 95°C for 90 min using a GFL1002 water-bath (GFL Company,
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Burgwedel, Germany). After being cooled to 37°C, the prepared RSM was inoculated with L. casei LC2W (5 × 106 CFU/g) and S. thermophilus ST1 (5 × 106 CFU/g). Cultures
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without phage were designated LC-ST, while those supplemented with S. thermophilus
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phage φ101 were designated LC-ST-P. RSM was inoculated with single L. casei LC2W (5
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× 106 CFU/g or 1 × 107 CFU/g) or S. thermophilus ST1 (5 × 106 CFU/g or 1 × 107 CFU/g) as a control (designated LC and ST). The samples of RSM fermented by L. casei LC2W
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(designated LC-P) or S. thermophilus ST1 (designated ST-P) in the presence of S. thermophilus phage were also cultured and analyzed. All of the fermented milk cultures were incubated at 37°C and were monitored automatically (recorded every 5 min) for pH using a Cinac system (Alliance Instruments, Mery-Sur-Oise, France) during milk acidification. The number of viable L. casei and S. thermophilus cells were selectively counted during fermentation using MRS (fermented milk sampled every 12 h for 72 h) and LM17 (fermented milk sampled every 2 h for 24 h) agar (Ashraf and Shah, 2011), respectively.
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2.3. Preparation of S. thermophilus CFS
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S. thermophilus ST1 was grown in 12% RSM at 37°C for 24 h. The fermented milk
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was centrifuged at 8000 × g for 10 min at 5°C. The supernatant was adjusted to a pH of 6.5 using 1 M NaOH and then filtered through a 0.22-μm pore-size filter (Millipore,
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Billerica, MA, USA) for sterilization. The CFS was lyophilized and kept frozen (-80°C)
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until use.
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2.4. S. thermophilus cell collection and production of CCE
S. thermophilus cell collection was carried out according to Gaudreau et al. (2005),
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and production of the CCE was carried out according to Tanigawa et al. (2010) with some modifications. A total of 10 L of sterile (110°C for 15 min) RSM (12%, w/w) was
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inoculated with S. thermophilus ST1 (1 × 107 CFU/g) and cultured at 37°C in a BLBIO-10SJ fermenter (Bailun, Shanghai, China). Agitation of the cultures was kept at 50 rpm, and the pH (maintained at 5.6) was buffered with 5 N ammonium hydroxide. Fermentations were stopped after 24 h of incubation. Hereafter, the viable S. thermophilus cells were counted using LM17 agar. Cell pellets were collected by centrifugation (8000 × g for 10 min at 5°C) and washed thrice in TMA-I buffer (10 mM Tris-HCl, pH 7.8, 30 mM NH4Cl, 10 mM MgCl2, and 6 mM 2-mercaptoethanol) in order to avoid the precipitation of casein during centrifugation. After washing twice in sterile saline (0.85%, w/w), the cell pellets were ground on four separate, consecutive occasions: beating for 20 s each at 3000 7
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rpm with 0.1-mm zirconia silica beads in a Biospec mini bead-beater-16 (Bartlesville, OK, USA), after removing the beads and cell debris by centrifugation, the cell lysates were
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adjusted to 5 × 109 ruptured S. thermophilus cells/g using distilled water according to the
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aforementioned S. thermophilus viable cell count. Then, lysates were filtered through a 0.22-μm pore-size filter for sterilization. The obtained CCE was kept frozen (-80°C) ready
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for use.
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2.5. Effect of S. thermophilus CFS and CCE on L. casei
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The growth-promoting properties of CFS and CCE isolated from S. thermophilus on L. casei LC2W were determined by evaluating the acidification performance and L. casei
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cell viability. Different concentrations of heated (sterilized at 110°C for 15 min) or native (sterilized using a filter) CFS and CCE were added to sterile RSM (final concentration of
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RSM, 12%, w/w) to prepare the media. The CFS- and CCE-supplemented media were then inoculated with L. casei LC2W (1 × 107 CFU/g) and incubated at 37°C. RSM without supplementation was inoculated with L. casei LC2W as a control. The pH of each culture was measured with a 420A pH-meter (Thermo Orion, Beverly, MA, USA) and the viable cells were counted using MRS agar every 24 h.
2.6. Statistical analysis
All physicochemical experiments were performed in triplicate, and the results are 8
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expressed as the mean or means ± standard deviation (SD). Significant differences among
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treatments were evaluated by performing analysis of variance (ANOVA) F tests and
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Fisher's least significant difference (p < 0.05) using Statgraphics Plus 5.1 software
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(Manugistics, Rockville, MD, USA).
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3. Results and discussion
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3.1. Acidification performance of L. casei co-cultured with S. thermophilus
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L. casei and S. thermophilus presented different acidification rates in milk(Fig. 1).
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The pH of the fermented milk rapidly decreased after inoculation with a single strain of S.
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thermophilus (ST). However, the acidification slowed down when the pH dropped to around 5.0, and completely halted at pH 4.4. The pH of the fermented milk inoculated
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with L. casei alone (LC) decreased slowly in the early fermentation stage, but fast acidification was observed after incubating for 6 h. Co-culture of L. casei and S. thermophilus (LC-ST) appeared to combine the advantages of the two strains. In these co-cultures, the pH of the inoculated fermented milk decreased constantly throughout the incubation, indicating that the dual inoculators had a higher level of acidification capability. In general, LAB such as S. thermophilus and L. bulgaricus possess strong β-galactosidase and protease activities and they are fast-acting acidifiers in milk (Shah and Jelen, 1991). Unfortunately, probiotic bacteria such as L. casei usually possess weak
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β-galactosidase,
phospho-β-galactosidase,
protease
activities
and
lactose
phospho-transferase system (Ikram ul and Mukhtar, 2006; Mozzi et al., 2009; Wu et al.,
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2009). Notably, phospho- and β-galactosidase, as well as the entire proteolytic system, are
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crucial for LAB and probiotic bacteria to grow in milk. These enzymes are usually located in the cell wall or intracellular compartment (Savijoki et al., 2006). Mixture of selected
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bacterial strains could be adopted to obtain desirable characteristics such as high probiotic viable cells, fast acidification, and good flavor. For the intrinsic intracellular proteolytic
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enzymes, it is possibly beneficial to lyse the cells to exploit them when co-cultured with probiotic bacteria. Therefore, a specific bacteriophage for S. thermophilus was
L. casei (LC-ST-P).
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supplemented to the co-cultures to investigate the interaction between S. thermophilus and
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The acidity produced by these bacteriophage-spiked co-cultures was slightly lower than that observed from the LC-ST co-cultures without phage during the first 24 h, which
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might be due to phage invasion that weakened the acidification ability of the S. thermophilus strain. Virulent S. thermophilus phage φ101was previously shown to be destructive to a corresponding sensitive strain during fermentation (Ma et al., 2014). In the present study, the pH of S. thermophilus with phage (ST-P) decreased during the first 9 h after inoculation. However, the acidification completely halted at pH 5.3 because of the reproduction and releasing of bacteriophages (Fig. 1). Interestingly, the decrease in pH of the LC-ST-P cultures surpassed that of the LC-ST cultures from 24 to 72 h, indicating an enhanced level of acidification. For this reason, we thought that phage invasion introduced the intracellular lysate of S. thermophilus into the media, which stimulated the growth and 10
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acidification ability of L. casei. The acidification curves of LC were mostly coincident
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with that of LC-P (samples fermented by L. casei in the presence of S. thermophilus phage,
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acidification curves not shown), which demonstrated that the phages of S. thermophilus
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had no effect on L. casei. Overall, the co-cultures especially in the presence of phage shortened the fermentation time. For example, the pH value of LC fermented milk was
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3.72 at 72 h. However, the fermentation times reaching the same pH end point (3.72) in LC-ST and LC-ST-P fermented milk were only 61 h and 40 h, respectively (Fig. 1). The
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acidification performance was promoted by L. casei co-cultured with phage-lysed S. thermophilus, which provided a feasible and effective method to improve probiotic
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fermentation.
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3.2. Growth and propagation of L. casei co-cultured with S. thermophilus
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The numbers of L. casei found in the LC, LC-ST, and LC-ST-P fermented milk cultures at 72 h were 8.83, 7.31, and 9.35 log CFU/g, respectively (Fig. 2). Compared to the LC fermented milk samples, a significant decrease of 1.52 log CFU/g was observed in the LC-ST fermented milk samples, indicating that L. casei cell viability was affected by co-cultured S. thermophilus during milk fermentation. It seemed that the probiotic L. casei was inhibited by the metabolites secreted by S. thermophilus (for example, organic acids and antimicrobial agents). Another possible explanation of this bacterial population changes was that the added S. thermophilus was competitive for the nutrients present in the milk. When the phage was added to the co-cultivation, the number of viable L. casei at 11
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72 h increased by 0.52 and 2.04 log CFU/g compared to the LC and LC-ST cultures,
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respectively (Fig. 2). It was consistent with the assumption that the decrease in the number
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of viable S. thermophilus bacteria caused by the phage invasion eliminated the competitive
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inhibition of L. casei in late fermentation, resulting in an increase in the viability of L. casei in the co-culture. Indeed, compared to LC, the slight increase in L. casei cell count in
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the LC-ST-P cultures indicated that lysing and releasing of the cellular contents of S. thermophilus by the virulent phage stimulated the growth and propagation of L. casei.
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These results were also consistent with the changes observed for the acidification performance, whereby the phage-supplemented co-cultures showed the strongest
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acidification ability.
S. thermophilus is a LAB that has the ability to quickly acidify milk. In fermented
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milk samples containing only S. thermophilus (ST), the bacteria entered the exponential growth phase after incubating for 2 h and the cells number of S. thermophilus peaked after
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12 h (Fig. 3). In the co-cultured experiments (LC-ST), the change in the number of S. thermophilus mirrored the changes observed when S. thermophilus was cultured alone. In the LC-ST-P samples, S. thermophilus grew and reproduced rapidly in the early stage of fermentation (2–8 h), as expected, and decreased very quickly in the later stages of fermentation following phage invasion (Fig. 3). The S. thermophilus cell count of ST-P fermented milk increased rapidly in the early stage of fermentation and decreased quickly in the later stages of fermentation (data not shown), which was analogous to the change observed in the ST-LC-P. Taken together, these results suggest that while the acidification performance of the 12
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co-culture LC-ST was significantly higher than that of the single LC or ST cultures, the
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probiotic content of L. casei was rather low, which indicated that simple co-cultivation
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was impractical to enhance the probiotic production in customer dairy products.
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Fortunately, the fastest acidification and the highest L. casei cell count were observed in the phage-supplemented co-culture experiments. This provided a straightforward approach
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to increase the probiotic content of this dairy product while simultaneously shortening the fermentation time. Furthermore, other probiotic strains such as Lactobacillus plantarum, L.
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rhamnosus, and Bifidobacterium sp. also showed stimulated growth and propagation when co-cultured with S. thermophilus spiked with specific phage (data not shown), which
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suggested that this co-culturing technique could be applied widely to promote various probiotic bacteria fermentation. Detailed investigation was needed for the large-scale
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application of this system in the food industry. Furthermore, discovering the mechanism of this enhanced L. casei probiotic content and acidification by the lysed S. thermophilus is
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essential to the implementation of this technique.
3.3. Acidification performance of L. casei supplemented with CFS and CCE
In order to elucidate the role of extracellular and intracellular metabolites and signaling molecules, the effects of the CFS and CCE isolated from S. thermophilus on the cultured L. casei bacteria were investigated. The CFS was known to contain the metabolites and secretory components utilized by S. thermophilus during milk fermentation. Therefore, determining the effects of isolated CFS on L. casei could reveal 13
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the interactions between L. casei and the extracellular metabolites of S. thermophilus. The
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CCE contains the lysed intracellular products of S. thermophilus cells. Determining the
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effect of CCE on L. casei could demonstrate the interactions occurring between L. casei
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and the intracellular metabolites of S. thermophilus in the presence of S. thermophilus phage. Thus, cracked and un-cracked, CCE and CFS of S. thermophilus were added into
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RSM during L. casei fermentation, and the acidification and propagation of L. casei were evaluated.
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As shown in Table 1, the addition of native CCE, at a final concentration of 0.2–2.0% (w/w), to the RSM-L. casei culture induced a statistically significant change in pH (p
0.05) were observed only adding 0.02% native CCE. Furthermore, heat greatly diminished the effect of CCE on the acidification performance in the L. casei cultures (for example, CCEN2.0% decreased pH by 2.10 vs. only 1.58 for CCEH2.0% at 24 h). These data suggested that a large proportion of the intracellular components isolated from S. thermophilus were heat-inactivated components such as biological active enzymes, while a smaller proportion were likely composed of non-temperature-dependent chemical molecules such as growth factors. In regards to the role of extracellular S. thermophilus molecules, no statistically significant differences (p > 0.05) in the acidification performance were observed following 14
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the addition of either native or heated CFS, at a final concentration of 0.02–2.0% (w/w) in
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12% RSM compared to the untreated control cultures (data not shown). These results are
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consistent with Vinderola et al. (2002), who also found that the CFS obtained from skim
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milk cultures fermented by S. thermophilus had no positive effect on probiotic L. casei.
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3.4. Growth and propagation of L. casei supplemented with CFS and CCE
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In addition to investigating the effect of CFS and CCE on the L. casei-induced acidification, we also studied the growth response of this bacterium to the CFS and CCE at
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different concentrations. 0.2–2.0% native CCE and 2.0% heated CCE isolated from S. thermophilus clearly enhanced the growth and propagation of L. casei in these
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experiments. The viable L. casei counts of CCEH2.0%, CCEN0.2%, and CCEN2.0% were 9.16, 9.28, and 9.40 log CFU/g, increase in cell viability at 0.33, 0.45, and 0.57 log CFU/g,
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compared to the control at 72 h (Table 1), respectively. These results are similar to those found for the phage-lysed S. thermophilus, whereby the growth and propagation of the co-cultured L. casei were enhanced. We suspect that the same components and signaling mechanism caused this change in viability after the addition of CCE. However, the addition of 0.02% native CCE (i.e., 106 ruptured S. thermophilus cells/g) showed no significant differences (p > 0.05). Therefore, the stimulatory growth and propagation on L. casei could be observed only when enough CCE was added
(more than 107 ruptured S.
thermophilus cells/g). Similar to the results from the acidification experiments, no statistically significant 15
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differences (p > 0.05) in cell viability were observed following the addition of native or
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heated CFS (data not shown). CFS is known to contain low levels of free amino acids and
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soluble peptides, which benefits for cell growth and viability (Serra et al., 2009; Vinderola
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et al., 2002). Zhang et al. (2010) showed that milk peptides could promote the growth of LAB. However, it is possible that the peptide content in the CFS isolated in this study was
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too low to stimulate L. casei effectively. Additional work is necessary to fully determine the role of the extracellular components of S. thermophilus CFS in L. casei.
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In brief, we showed that S. thermophilus could be used to compensate for the slow acid-producing ability of L. casei. S. thermophilus-specific bacteriophage, used to rupture
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viable S. thermophilus cells, was also shown to stimulate the growth and propagation of L. casei. Notably, a reduced fermentation time and high L. casei cell count were achieved
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during fermentation in the co-cultures. Although phages are considered to be unfavorable environmental factors and retard fermentation in yogurt and cheese manufacturing (Zinno
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et al., 2010), they played a unique and positive role in lysing their corresponding LAB and releasing the intracellular products during the production of fermented milk with high probiotic content. We believe that the addition of S. thermophilus to the probiotic-enriched milk can enhance the consumer product two-fold. First, S. thermophilus is a fast-acidifying strain in milk and compensates for the slow acid-producing ability of L. casei, especially in the early exponential growth phase, which helps to rapidly reduce the pH of the fermented milk and avoid the risk of undesirable microorganism growth. Second, L. casei is a fastidious microorganism (Cai et al., 2009; Du Toit et al., 2011) and the pool of free amino acids and peptides in milk is not sufficient to fulfill optimal bacterial growth. Thus, 16
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the phage-induced release of the S. thermophilus cell lysate, which contains proteases,
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lactase, free amino acids, and peptides, accelerates the growth and propagation of the
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probiotic. Probiotic growth is then further enhanced, as the decrease in viable S.
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thermophilus cells also eliminates the competition between this bacterium and the probiotic for nutrients in the media.
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This study is the first to investigate the relationship between co-cultured S. thermophilus and L. casei as well as the effects of S. thermophilus-derived CFS and CCE
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on the cell count and milk fermentation capabilities of L. casei. Research concerning probiotics, LAB/probiotic co-culture techniques, and dairy product fermentation is far
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from complete, and additional work to uncover the full relationship between LAB and
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probiotic bacteria is warranted.
Acknowledgements
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This work was supported by National High Technology Research and Development Program of China (2011AA100901), and the National Key Technologies Program of China (2013BAD18B01) during the 12th Five-Year Plan Period.
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Table:
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Table 1 The influence of CCE on pH values and viable L. casei counts of fermented milk
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Figure legends:
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Fig. 1 Acidification curves during fermentation using different starter cultures ST: Fermented by S. thermophilus; LC: Fermented by L. casei; LC-ST: L. casei
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co-culturing with S. thermophilus; LC-ST-P: L. casei co-culturing with S. thermophilus in the presence of S. thermophilus phages; ST-P: Fermented by S. thermophilus with S.
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thermophilus phages.
All the samples fermented by L. casei/S. thermophilus at total inoculum of 1×107 CFU/g
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The phage inoculum size was 1×102 PFU/g.
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Fig. 2 Changes in viable L. casei counts during milk fermentation LC: inoculated with single L. casei (5 × 106 CFU/g); LC-ST: inoculated with L. casei (5 ×
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106 CFU/g) and S. thermophilus (5 × 106 CFU/g); LC-ST-P: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g) in the presence of S. thermophilus phages (1×102 PFU/g).
Fig. 3 Changes in viable S. thermophilus counts during milk fermentation ST: inoculated with single S. thermophilus (5 × 106 CFU/g); LC-ST: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g); LC-ST-P: inoculated with L. casei (5 × 106 CFU/g) and S. thermophilus (5 × 106 CFU/g) in the presence of S. thermophilus phages (1×102 PFU/g). 23
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pH values
L. casei counts (log CFU/g)
Sampls 24 h
48 h
72 h
RSM
6.36±0.03a
4.98±0.06 a
3.86±0.05 a
3.74±0.03 a
8.83±0.16 b
CCEN0.02%
6.36±0.03 a
4.92±0.05 a
3.81±0.04 a
3.73±0.03 a
8.89±0.15 b
CCEN0.2%
6.36±0.05 a
4.61±0.06 c
3.69±0.06 b
3.59±0.02 b
9.28±0.10 a
CCEN2.0%
6.38±0.03 a
4.28±0.04 d
3.65±0.04 b
3.57±0.03 b
9.40±0.17 a
CCEH0.02%
6.35±0.03 a
4.99±0.05 a
3.87±0.07 a
3.75±0.03 a
8.82±0.12 b
CCEH0.2%
6.37±0.05 a
4.97±0.07 a
3.86±0.06 a
3.73±0.04 a
8.87±0.11 b
CCEH2.0%
6.38±0.04 a
4.80±0.05 b
3.71±0.05 b
3.62±0.03 b
9.16±0.14 a
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0h
RSM: fermented milk without supplementation (control); CCEN0.02%, CCEN0.2% and CCEN2.0%:
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fermented milk containing 0.02%, 0.2% and 2.0% native CCE; CCEH0.02%, CCEH0.2% and CCEH2.0%:
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fermented milk containing 0.02%, 0.2% and 2.0% heated CCE; The L. casei counts are the values measured at 72 h. All values are mean ± SD (n=3). Rows with different superscript letters indicate
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statistical differences (P < 0.05).
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Highlights
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1. S. thermophilus compensated for the slow acid production of L. casei.
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2. Phage-induced lysis eliminated the competitive inhibition on L. casei.
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3. The lysate of S. thermophilus stimulated the growth and propagation of L. casei.
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4. The effect of CCE on L. casei confirmed the stimulatory role of the phage.
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