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Lipase inhibitory activity of skim milk fermented with different strains of lactic acid bacteria
Journal of Functional Foods 60 (2019) 103413
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Lipase inhibitory activity of skim milk fermented with different strains of lactic acid bacteria Ana María Gil-Rodríguez, Thomas P. Beresford
T
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Teagasc Food Research Centre, Moorepark and Food for Health Ireland, Fermoy, Co. Cork P61C991, Ireland
A R T I C LE I N FO
A B S T R A C T
Keywords: Obesity Lactic acid bacteria Lipase inhibitory activity Fermented milk
One of the main causes of obesity is an imbalance between energy intake and expenditure. One approach to facilitate weight loss is to decrease absorption of energy-rich nutrients by inhibition of pancreatic lipase. A spectrophotometric method was developed to evaluate the potential of milk fermented with lactic acid bacteria to inhibit pancreatic lipase in vitro. Fermentates produced with Lactobacillus helveticus strains at 42 °C exhibited the highest levels of lipase inhibition, with strains SC8, SC44 and SC45 inhibiting pancreatic lipase by > 49%, significantly higher than the activity of a milk control. In all cases activity was retained after removal of large proteins including casein and in the < 3 kDa fraction, but not in the 3–10 kDa fraction. Although these results require validation with in vivo models, the strains L. helveticus SC8, SC44 and SC45 are good candidates for use in the production of functional food products with potential anti-obesity effects.
1. Introduction Obesity is an increasing health problem with rising incidence rates, not only in industrialised countries, but also in developing countries. It is usually associated with other pathologic conditions, such as type 2 diabetes mellitus, hypertension, obstructive sleep apnoea and cardiovascular disease (Chen, He, & Huang, 2014; Marrelli, Loizzo, Nicoletti, Menichini, & Conforti, 2014). Apart from appetite suppression, one of the most important therapeutic strategies in the treatment of obesity is the administration of digestion and absorption inhibitors in order to decrease energy intake from the diet. As two major energy sources, the reduction in the absorption of triglycerides and glucose are usually attempted (Marrelli et al., 2014). The main source of dietary fat is triacylglycerol, but this molecule cannot be absorbed in the gut without being hydrolysed into smaller molecules such as monoglycerides and free fatty acids (Tucci, Boyland, & Halford, 2010). Pancreatic lipase is the enzyme responsible for this hydrolysis, subsequently facilitating the absorption of dietary fat. As excessive ingestion of fat is directly related with overweight and obesity, inhibition of this enzyme is a relevant approach for weight loss in addition to reduced-fat diets (de la Garza, Milagro, Boque, Campión, & Martínez, 2011). However, there is only one pharmacologic treatment (Orlistat) currently available to inhibit this enzyme and consequently other inhibitors are being sought (Kang & Park, 2012; Marrelli et al.,
2014; Tucci et al., 2010). Orlistat (tetrahydrolipstatin) is a lipase inhibitor derived from lipstatin, a natural product obtained from the bacteria Streptomyces toxytricini (Borgström, 1988). It inhibits the absorption of fat from the diet by 30%; thus, facilitating weight loss (Kang & Park, 2012). As a result of side effects caused by orlistat (e.g. abdominal pain, diarrhoea, oily stools, liposoluble vitamin deficiencies) and other digestive enzyme inhibitors, new sources are currently being screened in search for milder inhibitors without side effects (Buchholz & Melzig, 2016; Tucci et al., 2010). In this context, the search for lipase inhibitors has focused mostly on herbs and edible plants, but also fungi and bacteria from diverse origins (Reviewed in de la Garza et al., 2011). Apart from reduced side effects, lipase inhibitors naturally present in food products could have the advantage of reducing body weight in obese subjects without the administration of drugs or dietary supplements. Fermented foods (especially fermented dairy products) have traditionally been associated with multiple health benefits. The anti-obesity effect of kefir (Kim et al., 2017; Lim et al., 2017) and milk fermented or supplemented with lactobacilli (Ogawa, Kobayashi, Sakai, Kadooka, & Kawasaki, 2015; Park, Seong, & Lim, 2016; Pothuraju et al., 2016; Sato et al., 2008) has been demonstrated in overweight mice, rats and humans. These (and other) health benefits of fermented dairy products have been linked to milk-derived bioactive peptides (Aguilar-Toalá et al., 2017), which can be released from the native protein through
Abbreviations: NFSM, non-fermented skim milk; RSM, reconstituted skim milk; LAB, lactic acid bacteria; NPC, 4-nitrophenyl octanoate; DMSO, dimethyl sulfoxide ⁎ Corresponding author. E-mail addresses: [email protected] (A.M. Gil-Rodríguez), [email protected] (T.P. Beresford). https://doi.org/10.1016/j.jff.2019.06.015 Received 5 June 2018; Received in revised form 16 May 2019; Accepted 10 June 2019 Available online 16 July 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
2.3. Reconstituted skim milk (RSM) preparation
different mechanisms, one of them being the action of microbial proteolytic enzymes (Qian et al., 2011). In addition, the ability of different strains of lactic acid bacteria (mainly lactobacilli) and bifidobacteria to inhibit pancreatic lipase has been demonstrated in several studies using reconstituted freeze-dried cells (An et al., 2011; Ogawa et al., 2015; Park, Cho, Kim, & Lim, 2014; Zhou et al., 2013). Similarly, some strains of Lactobacillus curvatus, Lactobacillus plantarum and Weisella koreensis have been shown to reduce lipid accumulation and adipogenesis in preadipocyte 3T3-L1 cells (Moon et al., 2012; Park, Ahn, Huh, Jeon, & Choi, 2011). In this study we investigated the potential of milk fermented with different strains of lactic acid bacteria (LAB) from the genera Lactobacillus, Lactococcus and Pediococcus to inhibit pancreatic lipase activity in vitro. In addition, as fermentation temperature has an impact on metabolic activity (including proteolysis) and other parameters such as growth rate of LAB (Khanal & Lucey, 2018; Savijoki, Ingmer, & Varmanen, 2006), we investigated the effect of fermentation temperature in the development of this beneficial activity. Furthermore, we analysed the water soluble fraction following centrifugation of the samples with highest activities and their low molecular size fractions (< 10 kDa, 3–10 kDa and < 3 kDa) in order to determine the molecular range size of the active compounds.
Skim milk powder was reconstituted at 10% (w/v) by slowly adding the powder into distilled water with stirring. Once all the powder was incorporated the milk was left to rehydrate for 2 h with constant stirring. The RSM was sterilised by heat treatment (121 °C, 5 min) to inactivate indigenous microbes, followed by cooling and overnight storage at 4 °C. The milk was then distributed into the fermentation containers and incubated for 1 h at the appropriate fermentation temperature before inoculation with the strain of interest. 2.4. Milk fermentation Fresh overnight cultures (24 h) as described above were used to inoculate (1% v/v) heat-treated 10% RSM. Fermentations were performed at four different temperatures (30, 33, 37 and 42 °C) for each microbial strain and were run for 24 h under aerobic conditions without agitation. During milk fermentation by most LAB lactose is metabolised with production of lactic acid as the main by-product (Gänzle, 2015). Therefore; the pH of the product was measured at the end of the fermentation as an indicator of strain performance in milk. 2.5. Sample fractionation
2. Materials and methods
The samples with the highest lipase-inhibitory activities were subject to fractionation by molecular size in order to isolate the fractions potentially containing bioactive peptides (< 10 kDa) [Aguilar-Toalá et al., 2017, Giacometti & Buretić-Tomljanović, 2017]. The samples were centrifuged (4000 rpm, 30 min) to remove large proteins including casein and the supernatants were sterilised by filtering through a 0.2 µm pore size Polyethersulfone (PES) filters. An aliquot of the resulting filtrate was stored at −20 °C for analysis, and the rest was ultrafiltered in succession using centrifugal filters with different molecular weight cut-offs. First they were pre-filtered through 50 and 30 kDa molecular weight cut-off centrifugal filters in order to remove the molecules with highest molecular weights. The 30 kDa permeate was then filtered through 10 and 3 kDa molecular weight cut-off filters and the fractions < 10 kDa, 3–10 kDa and < 3 kDa were filter-sterilised using a 0.2 µm pore size PES filters and stored at −20 °C for analysis. A control of non-fermented skim milk (NFSM) was also included for reference. In order to separate the supernatant from the control milk the casein was precipitated by acidifying the milk using 0.1 N HCl until a pH of 4.6 was reached (Aguilar-Toalá et al., 2017). After acidification, the control milk was centrifuged under the same conditions as the fermented milk samples and the supernatant was recovered and treated as outlined above.
2.1. Chemicals The anaerobic atmosphere generator Anaerocult A was purchased from OCON Chemicals (Cork, Ireland). De Man, Rogosa, Sharpe (MRS) and M17 culture media were obtained from BD (Oxford, UK). Amicon centrifugal filtration units for sample fractionation were purchased from Merck Millipore (Cork, Ireland). All other reagents used in this study were purchased from Sigma Aldrich (Arklow, Ireland).
2.2. Microbial strains and culture conditions In this study, 31 strains of LAB belonging to the DPC culture collection (Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland) were used. These strains were selected among 7 different species of the genera Lactobacillus, Lactococcus and Pediococcus (Table 1) with QPS (qualified presumption of safety) status. The strains of Lactobacillus and Pediococcus were grown in MRS supplemented with 0.5 g/L L-cystine hydrochloride and incubated at 37 °C under anaerobic conditions generated using Anaerocult A, whereas Lactococcus strains were grown at 30 °C in M17 supplemented with 10 g/L α-lactose. Before milk fermentation each strain was transferred from a frozen glycerol stock culture maintained at −20 °C onto an agar plate and incubated 48 h. A single colony was isolated onto a fresh agar plate to ensure culture purity. From this second plate a liquid culture was inoculated. This fresh overnight culture (24 h) was in turn used to seed (1% v/v) a second broth that would be used to inoculate the milk.
2.6. Peptidic profile The peptidic profile of the samples with highest lipase inhibitory activity, their supernatants and the active fractions (< 3 kDa) were obtained as described by Slattery and Fitzgerald (1998). Briefly, 200 µL of each sample were diluted in 9.8 mL of distilled water and filtered through a 0.2 µm Polyvinylidene Fluoride (PVDF) filter. The dilutions were subsequently analysed by size exclusion chromatography (SEC) using a TSK G2000SW (300 × 7.5 mm) and a TSK G2000swxl column (300 × 7.8 mm, Tosu Hass, Japan) in series, fitted to a Waters Alliance 2695 separation module (Waters Corporation, Milford, Mass, USA). The mobile phase used was prepared by adding HPLC grade acetonitrile (30% v/v) and trifluoroacetic acid (0.1% v/v) to R2 filtered water. Assays were run using a flowrate of 1 mL/min and continually monitored at 214 nm using a Waters 2487 dual wavelength detector. Chromatographic data were collected and analysed using Empower data handling software package (Waters Corporation, Milford, MA, USA). Standard proteins and peptides were used to prepare a retention time to molecular weight calibration curve.
Table 1 Species of LAB and number of strains per species used in this study. Species
No. strains
Lactobacillus acidophilus Lactobacillus brevis Lactobacillus delbrueckii ssp. bulgaricus Lactobacillus helveticus Lactobacillus plantarum Lactococcus lactis Pediococcus pentosaceus
4 2 2 13 4 3 3
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A.M. Gil-Rodríguez and T.P. Beresford
Table 2 Milk pH values after 24 h fermentation at different temperatures. Species
Strain no.
pH 30 °C
L. acidophilus
L. brevis L. delbrueckii ssp bulgaricus L. helveticus
L. plantarum
Lc. lactis
P. pentosaceus
SC1 SC2 SC3 SC63 SC37 SC92 SC40 SC77 SC4 SC5 SC6 SC7 SC8 SC41 SC42 SC43 SC44 SC45 SC46 SC47 SC64 SC70 SC71 SC72 SC80 SC15 SC18 SC34 SC29 SC30 SC57
6.11 5.88 5.88 5.76 6.39 6.43 4.40 4.25 3.95 5.13 5.00 4.09 4.86 4.75 4.79 4.84 5.01 4.82 5.72 4.64 5.46 4.89 4.94 4.92 5.20 4.28 4.25 4.26 6.04 6.04 6.12
33 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.09 0.03 0.09 0.00 0.01 0.20 0.13 0.07 0.03 0.12 0.01 0.08 0.30 0.14 0.04 0.05 0.10 0.11 0.05 0.08 0.04 0.06 0.04 0.06 0.01 0.00 0.01 0.12 0.06 0.00
5.54 5.22 5.15 5.24 6.39 6.34 3.76 3.72 3.66 4.56 4.01 3.72 3.97 3.97 3.93 3.96 3.96 3.88 5.37 3.72 5.00 4.75 4.75 4.75 5.12 4.38 4.33 4.31 5.93 5.89 6.09
37 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.00 0.04 0.04 0.06 0.00 0.16 0.12 0.03 0.04 0.11 0.03 0.13 0.21 0.06 0.12 0.09 0.00 0.12 0.03 0.04 0.08 0.06 0.07 0.08 0.04 0.04 0.03 0.16 0.07 0.05
5.47 4.83 4.87 4.83 6.49 6.47 3.92 3.74 3.57 3.95 3.69 3.68 3.73 3.59 3.61 3.60 3.60 3.60 5.24 3.58 4.60 4.67 4.70 4.70 5.09 4.49 4.44 4.43 5.94 5.87 6.03
42 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.43 0.47 0.04 0.07 0.06 0.17 0.12 0.08 0.01 0.17 0.18 0.23 0.13 0.10 0.10 0.12 0.10 0.02 0.05 0.04 0.04 0.07 0.06 0.12 0.00 0.05 0.04 0.16 0.07 0.01
4.79 5.00 4.91 4.87 6.52 6.53 3.75 3.72 3.59 3.84 3.61 3.53 3.46 3.48 3.53 3.65 3.42 3.43 5.04 3.41 4.21 5.58 5.62 5.61 5.79 6.25 5.03 5.01 5.98 6.37 6.07
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.10 0.04 0.06 0.10 0.08 0.05 0.21 0.19 0.11 0.26 0.11 0.09 0.07 0.10 0.21 0.02 0.02 0.10 0.02 0.16 0.31 0.42 0.44 0.05 0.19 0.16 0.20 0.02 0.04 0.09
measurements. Apart from eliminating the turbidity due to the addition of milk to the reaction mixture, the clarification step also stops the enzymatic reaction between lipase and NPC due to a pH change (Humbert, Guingamp, Linden, & Gaillard, 2006). The mixture was gently mixed and subsequently incubated 3 min at 37 °C. After incubation, absorbance was measured at 412 nm using a Jenway 6300 Spectrophotometer (Cole-Parmer Ltd, Staffordshire, UK). Due to the increase in the buffer concentration, turbidity still remained in some of the samples. These samples were filtered using a 0.2 µm PES filter before absorbance measurement. The percentage of lipase inhibitory activity was calculated with respect to 100% activity reference control as follows:
2.7. Lipase inhibitory activity test For the detection of lipase inhibitory activity in the milk fermentates, a method was developed by adapting the protocols described by Bendicho, Trigueros, Hernández, and Martin (2001), Conforti et al. (2012) and Marrelli et al. (2014) with modifications. The pH of the reaction mixture is critical for the hydrolysis of 4nitrophenyl octanoate (NPC) into octanoate and 4-nitrophenol (yellow coloured product) by pancreatic lipase to proceed. The concentration of the buffers described in the cited literature (Tris-HCl between 0.05 and 0.07 M) were not sufficient to maintain the pH of the reaction at 8.5 after addition of the most acidic milk samples (pH ≤ 4.5). In order to maintain the pH in the optimal range for lipase activity under these conditions (8.25–8.75), increasing concentrations of Tris-HCl were tested. It was observed that at concentrations of Tris-HCl ≥ 0.15 M, the clarifying reagent for dairy products was unable to eliminate the turbidity of the samples (data not shown) and thus, a two buffer system was developed. For samples with pH > 4.5 and the reference control, Tris-HCl 0.10 M at pH 8.5 was used, whereas for samples with pH ≤ 4.5, Tris-HCl 0.10 M buffer was prepared at pH 9.5. To test the lipase inhibitory activity of the different fermented milks, 500 µL of sample were mixed with 2 mL of Tris-HCl buffer, following which 50 µL of a 5 mM solution of NPC in dimethyl sulfoxide (DMSO) were added. The reaction was initiated by adding 50 µL of a 5 mg/mL solution of pancreatic lipase (Type II, from porcine pancreas) dissolved in Tris-HCl buffer pH 8.5. The mixture was vortexed for 2 min at medium speed to avoid the formation of foam and incubated at 37 °C for 30 min. For the reference control, 500 µL of Tris-HCl buffer were added instead of sample. A blank was prepared for each sample, where NPC was substituted by 50 µL DMSO. A control using orlistat in DMSO (10 µg/mL) was included for comparison. After 30 min incubation at 37 °C, 1 mL of Clarifying Reagent for Dairy Products was added to each tube to allow absorbance
AbsS − AbsSB × 100⎞ Lipase inhibitory activity (%) = 100 − ⎛ ⎝ AbsRC − AbsB ⎠ ⎜
⎟
where AbsS is the absorbance of the sample, AbsSB is the absorbance of the corresponding blank, AbsRC is the absorbance of the reference control and AbsB is the absorbance of the blank of the reference control. The presence of milk in the reaction mixture provokes a reduction in the absorbance of p-nitrophenol of 5.71%. This value was experimentally obtained (data not shown) and applied as a correction factor to the net absorbance values before calculating the percentages of inhibition.
2.8. Statistical analysis Each milk fermentate was produced three times and analysed twice in duplicates. The results are expressed as average ± standard deviation. Significant differences were analysed with Pearson's chi-squared test. Values of P < 0.01 were considered statistically significant.
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3. Results
However, for strains of other species the opposite trend is observed, e.g., two out of the three Lc. lactis strains tested (SC18 and SC34) exhibited levels of lipase inhibitory activity significantly different from the control (35.44 ± 2.62% for SC18 and 38.29 ± 2.26% for SC34) when the fermentation was performed at 42 °C, but not when performed at lower temperatures (Fig. 1d). Contrarily as to what was observed for L. helveticus strains, for Lc. lactis SC18 and SC34 the lowest pH is reached when fermentation takes place at 30 °C (4.25 ± 0.00 for SC18 and 4.26 ± 0.01 for SC34) and increases progressively until it reaches its maximum at 42 °C (5.03 ± 0.16 for SC18 and 5.01 ± 0.20 for SC34), thus indicating a decreasing production of lactic acid as temperature increases, as would be expected (the optimum growth temperature for Lc. lactis is ∼30 °C). It is noteworthy that for this species, the maximum lipase inhibitory activity seems to coincide with the lower performance in milk and decreases together with pH. The strain Lc. lactis SC15 shows a trend similar to SC18 and SC34, but none of the activities registered for this strain are significantly different from the control. The other strains analysed, belonging to species L. acidophilus, L. brevis, L. delbrueckii ssp bulgaricus, L. plantarum and P. pentosaceus show moderate maximum activities with values around 35% inhibition (Fig. 1). L. acidophilus SC1 and SC63 show a similar profile, with higher activities at 37 and 42 °C, whereas SC2 and SC3 have their maximum at 33 °C. The two strains of L. brevis and the three strains of P. pentosaceus tested show no notable differences in pH at different fermentation temperatures, but some of the strains exhibit peaks of moderate activity at specific temperatures (e.g., 33 °C for P. pentosaceus SC29 and 37 °C for L. brevis SC37 and P. pentosaceus SC30). All strains of L. plantarum, except SC80, display a similar tendency to L. helveticus SC46 and SC64 with sequential increases in lipase inhibitory activity with rising fermentation temperatures until a maximum activity is reached at 37 °C. The strains L. plantarum SC70, SC71 and 72 show very similar profiles, although the activities obtained for strain SC70 are higher (maximum activity 36.83 ± 3.77% at 37 °C) than those obtained for SC71 and SC72. Interestingly, in all L. plantarum strains the pH values of the fermented milks decrease sequentially between 30 and 37 °C, but increase again at 42 °C, where lipase inhibitory activity decreases. Contrarily to what was observed for other strains of this species, inhibition levels by strain L. plantarum SC80 do not vary with temperature. Apart from SC80, this apparent lack of influence of fermentation temperature on lipase inhibitory activity is observed only with P. pentosaceus SC57. Although some strains from the same species show similar levels of lipase inhibition in their milk fermented products at a given temperature, the potential to inhibit pancreatic lipase seems strain-dependent (e.g., L. helveticus, Lc. lactis). Also, our data suggest that changes in the metabolic activity of LAB provoked by differences in fermentation temperature can strongly influence the development of lipase inhibitory activity (and potentially other bioactivities) during milk fermentation.
3.1. Strain performance in milk The reduction in pH with respect to NFSM (6.47 ± 0.03) was measured as an indicator of strain performance. Most of the strains used in this study reduced the pH of milk to some extent after 24 h fermentation at different temperatures, with Lactobacillus brevis strains as only exception (pH values between 6.34 and 6.53). Similarly, the pH reduction observed in milk fermented with Pediococcus pentosaceus was low, with values between 5.87 and 6.12 (Table 2). Generally, strains of the same species resulted in similar pH values after fermentation at a given temperature. However, some strains e.g., Lactobacillus helveticus SC46 and SC64 and L. plantarum SC80 at all temperatures and Lactococcus lactis SC15 at 42 °C all resulted in lower pH reductions to other strains of the same species under identical fermentation conditions. The lowest pH values (< 4) were registered for L. helveticus strains at 42 °C, increasing progressively as fermentation temperature decreased but staying below 4 at 37 and 33 °C for most strains. These were closely followed by Lactobacillus delbrueckii ssp bulgaricus strains with pH values below 4 for fermentates produced at 42, 37 and 33 °C. The other strains used in this study provoked more moderate decreases in pH. Lc. lactis strains acidified milk to values between 4.25 and 4.5 at 30, 33 and 37 °C, with the lowest values at 30 °C increasing progressively with temperature. Contrarily, milk fermented with Lactobacillus acidophilus and L. plantarum strains had their minimum pH value at 37 °C, increasing progressively as fermentation temperature decreased. 3.2. Development of lipase inhibitory activity during milk fermentation Under these experimental conditions (see Section 2.7 of Materials and methods), 10 µg/mL orlistat inhibited pancreatic lipase by 70.17 ± 4.61%. The highest levels of lipase inhibitory activity reported in this study were obtained for milk fermented with different strains of L. helveticus at 42 °C, with 8 products displaying inhibition levels above 45% (SC6, SC7, SC8, SC41, SC42, SC43, SC44 and SC45). Of these products, the highest activities were registered for strains SC8 (49.30 ± 3.56%), SC44 (49.43 ± 2.39%) and SC45 (49.75 ± 2.13%) (Fig. 1a). Overall, most of the milks fermented with L. helveticus strains developed high levels of lipase inhibitory activity at all tested temperatures, with some exceptions at 30 and 33 °C. The only milks whose levels of lipase inhibition did not reach 35% in any of the test conditions were those fermented with SC5, SC46 and SC64. In two of these (SC46 and SC64) activity increased with fermentation temperature until moderate levels were reached at 37 °C, but the activity decreased when fermentation temperature was further increased to 42 °C. Interestingly, the pH of the milk fermented by these two strains decreased with increasing temperature, having its highest value at 30 °C and the lowest at 42 °C (Table 2). As lactose fermentation leads to the formation of lactic acid and a subsequent decrease in pH, a lower pH value can be indicative of a higher level of metabolic activity and/or microbial growth. However, the pH values that these two strains attain after fermentation at 42 °C (5.04 ± 0.10 for SC46 and 4.21 ± 0.16 for SC64) are markedly different from the pH that the other strains of this species attained at this temperature (between 3.41 and 3.84). This suggests that these strains are metabolically different from the other strains analysed, thus possibly explaining the differences in lipase inhibitory activity observed between these two strains and the others from the same species. This could indicate that, for this species, the maximum activity of each strain is usually obtained in the product whose pH is lower, thus suggesting that metabolic activity may have an influence on the development of lipase inhibitory activity. This tendency can also be observed in strains of other species, such as L. delbrueckii ssp bulgaricus SC40 and SC77 (Fig. 1b) and L. acidophilus SC63 (Fig. 1c).
3.3. Analysis of water soluble fractions The three samples with highest lipase inhibitory activity (i.e., milk fermented by L. helveticus SC8, SC44 and SC45 at 42 °C) were selected for fractionation as described in Materials and methods. In summary large proteins (mostly caseins) were removed by centrifugation and the supernatants were filtered through a range of molecular weight cut-off filters resulting in fractions < 10 kDa, 3–10 kDa and < 3 kDa. As shown in Fig. 2, lipase inhibitory activity is retained in the supernatant from all three samples. All the samples analysed showed a markedly higher activity than the supernatant from NFSM with statistically significant differences. A notable reduction in the background activity of NFSM can be observed when casein is removed, as demonstrated in the reduction from 24.43 ± 1.20% to 2.54 ± 1.57% recorded in the NFSM samples for fermented milk and supernatant respectively. A reduction in lipase inhibitory activity was also noted for 4
Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
Fig. 1. Lipase inhibitory activity of milk fermented with different species of LAB at different temperatures (30, 33, 37 and 42 °C). The value obtained for the NFSM control is indicated as a horizontal bar, * indicates a significant difference between that sample and the NFSM control.
Interestingly, the activity observed after analysing the < 10 kDa fraction of all samples can be observed in the < 3 kDa fraction but not in the 3–10 kDa fractions, thus indicating that the activity observed is produced by a molecule with molecular size < 3 kDa.
the three supernatants relative to their respective fermented milk, further supporting the suggestion that the background activity in NFSM is mostly due to the presence of casein. The < 10 kDa fractions of all three samples exhibit levels of lipase inhibitory activity comparable to the levels observed in their respective supernatants before fractionation, albeit slightly reduced. This reduction; however, is only statistically significant in the sample SC8. As bigger proteins and molecules are removed during the ultrafiltration process, these results indicate that the inhibition of pancreatic lipase observed in these samples is produced by a medium or small sized molecule (below 10 kDa molecular weight), possibly a peptide released by hydrolysis of milk proteins by microbial proteolytic enzymes.
3.4. Peptidic profiles Peptidic profiles were obtained by size exclusion chromatography (SEC) as described in the materials and methods section and are presented in Fig. 3. It can be observed that the profiles of the three analysed fermentates and their associated supernatants and < 3 kDa fractions are very similar, suggesting similar proteolytic activities in the Fig. 2. Lipase inhibitory activity of the fermentates with the highest activities (L. helveticus SC8, SC44 and SC45 at 42 °C) and their respective fractions. a above the bar indicates a significant difference between that sample and the NFSM control. For comparison between the supernatants and small molecular size fractions (< 10 kDa, 3–10 kDa and < 3 kDa) of each fermentate, b indicates a statistically significant difference between that fraction and its respective supernatant prior to fractionation.
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Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
Fig. 3. Peptidic profiles of the fermentates with the highest activities (L. helveticus SC8, SC44 and SC45 at 42 °C) and their respective supernatants and < 3 kDa fractions: (a) SC8, (b) SC44, (c) SC45 and (d) NFSM control. The retention times highlighted in the profiles are, from left to right 12.181 min (molecules > 20 kDa), 14.979 min (20–10 kDa), 15.000 min (10–5 kDa), 18.000 min (5–2 kDa), 19.169 min (2–1 kDa), 20.226 min (1–0.5 kDa) and 24.596 min (< 0.5 kDa).
mw < 2 kDa (Fig. 3a–c). This suggests that milk proteins are completely hydrolysed by these strains into smaller peptides. As corresponds to their low molecular size, these two peaks can also be observed in the < 3 kDa fraction, which exhibited high lipase inhibitory activity in all cases. This suggests that either (a) one of the two peaks represents the active component or (b) both components are necessary to inhibit pancreatic lipase.
three strains used to produce these samples. In two of the NFSM profiles (intact sample and supernatant), two peaks can be observed (Fig. 3d), one in the area corresponding to molecules of mw > 20 kDa, which could correspond to caseins (23 kDa for β-casein and 24–26 kDa for α-casein), and a smaller peak in the area of 10–20 kDa, which could correspond to β-lactoglobulin, α-lactalbumin (18 kDa and 14 kDa, respectively), and κ-casein (19 kDa) (Vincent et al., 2016). In contrast, all the profiles of fermented milk samples (SC8, SC44 and SC45) and their fractions do not exhibit these peaks, but present two peaks in the area corresponding to molecules of
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A.M. Gil-Rodríguez and T.P. Beresford
4. Discussion
effect on overweight, obesity and related conditions. However, the mechanisms whereby these beneficial effects are achieved need to be further elucidated.
The anti-obesity effect of different species of LAB (mainly lactobacilli) and bifidobacteria has been reported in many studies, but only a few have investigated the potential of these beneficial microorganisms to inhibit pancreatic lipase in vitro (Ogawa et al., 2015; Park et al., 2014; Zhou et al., 2013) and, in all cases, the studies were performed on resuspended freeze-dried bacterial cells from washed cultures. In a similar study, peptidic fractions obtained by enzymatic hydrolysis of camel milk whey were reported to exhibit pancreatic lipase inhibitory activity levels between 25 and 55% (Jafar, Kamal, Mudgil, Hassan, & Maqsood, 2018). To our knowledge, the current study is the first where the potential anti-obesity effect of milk fermented with different strains of LAB and their derived low molecular size fractions has been assessed in vitro. The activities reported in the present study are moderate when compared to orlistat at 10 µg/mL (70.17 ± 4.61%). In line with these results, Park et al. (2014) reported that L. plantarum Q180 inhibits pancreatic lipase by 83.61 ± 2.31% at concentrations of 100 µg/mL by directly inhibiting the hydrolysis of p-nitrophenylpalmitate. Comparison of inhibition levels between the two studies is difficult; however, as the methods for lipase inhibition assessment are notably different despite being based on the release of a coloured product resulting from enzymatic hydrolysis. Furthermore, the activity is measured in different types of sample (resuspended lyophilised bacterial cells vs. fermented milk and water soluble fractions after casein removal). The mechanism whereby lipase inhibition is achieved in these fermented milk products is unclear and several mechanisms may be involved. In a recent study, the capacity of Lactobacillus gasseri LG2055 to inhibit pancreatic lipase in vitro was tested with two different methods: i) hydrolysis of 4-methylumbelliferyl oleate and ii) decrease in fat droplet size. In contrast to our results, the data obtained by these authors indicate that the activity of this strain is not due to a direct inhibition of the enzyme (no decrease in the hydrolysis of 4-methylumbelliferyl oleate is observed) but due to an increase in fat droplet size. This results in a decrease in the oil-water interface surface, which subsequently hinders lipase activity. These findings were related to a significant increase in faecal fat excretion in healthy adults after administration of milk fermented with this strain (Ogawa et al., 2015). Zhou et al. (2013) demonstrated that Lactobacillus pentosus Z-PT84 inhibited the hydrolysis of high-oleic safflower oil by pancreatic lipase. The authors hypothesised that the hydrophobicity of the cell surface may facilitate adhesion of the bacterial cell to the oil-water interface (where pancreatic lipase exerts its activity) and modify its mechanical properties, thus affecting lipase activity. In this context; the more hydrophobic the cell surface, the higher the potential for adhesion to the substrate interface and therefore, the higher the interference with lipase activity. In the present study the activity of the selected samples is retained after removal of casein and microbial cells during the filtration and fractionation process. Furthermore, the activity is retained in the < 3 kDa fraction in all three samples tested, which contained two groups of low molecular weight peptides (< 2 kDa), as demonstrated in the two peaks observed in the SEC profiles of all three samples. This suggests that in this case lipase is inhibited by a small size milk-derived peptide released during microbial fermentation. In line with our results, other studies have reported diverse bioactivities in the < 3 kDa fractions of milk fermented with different strains of LAB, and generally the activity of these fractions was higher or at least equal to the activity of the 3–10 kDa fractions (Aguilar-Toalá et al., 2017; Qian et al., 2011). In addition, peptides from milk kefir have recently been shown to modulate lipid metabolism in vivo by regulating the genetic expression of different enzymes involved in both lipogenesis and lipid oxidation in the liver (Tung et al., 2018). Collectively, these results present strong evidence of the potential of milk-derived bioactive peptides released by microbial fermentation to regulate lipid metabolism at different stages and exert a beneficial
5. Conclusions Our results indicate that different strains of LAB have the ability to produce fermented milks with the capacity to partially inhibit pancreatic lipase in vitro. Of all the products tested, milk fermented with L. helveticus SC8, SC44 and SC45 at 42 °C are good candidates as functional foods for weight loss and improvement of obesity-related pathologic conditions with reduced side effects. However, further studies are needed to evaluate the anti-obesity potential of these milk fermented products in appropriate in vivo models. The lipase inhibition activity associated with these fermented milks were observed in filter sterilised fractions containing low molecular weight molecules (< 3 kDa). Furthermore, these fractions exhibit two peaks corresponding to small size (< 2 kDa) peptides. These data suggest that the active component is a peptide released from a milk protein hydrolysed during fermentation by L. helveticus. The data reported here were generated using an in vitro assay for pancreatic lipase activity suggesting that the mechanism of action relates to inhibition of the enzyme, possibly by inhibition of the its active site or interference with a critical location on the enzyme. In addition, the method described in this paper could be considered as a first-step screening for fermented dairy products with potential anti-obesity activity. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgements This work was supported by Food for Health Ireland (Enterprise Ireland Grant: CC20080001). References Aguilar-Toalá, J. E., Santiago-López, L., Peres, C. M., Peres, C., Garcia, H. S., VallejoCordoba, B., González-Córdova, A. F., & Hernández-Mendoza, A. (2017). Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains. Journal of Dairy Science, 100(1), 65–75. An, H. M., Park, S. Y., Lee, D. K., Kim, J. R., Cha, M. K., Lee, S. W., Lim, H. T., Kim, K. J., & Ha, N. J. (2011). Antiobesity and lipid-lowering effects of Bifidobacterium spp. in high fat diet-induced obese rats. Lipids in Health and Disease, 10, 116. Bendicho, S., Trigueros, M. C., Hernández, T., & Martin, O. (2001). Validation and comparison of analytical methods based on the release of p-nitrophenol to determine lipase activity in milk. Journal of Dairy Science, 84(7), 1590–1596. Borgström, B. (1988). Mode of action of tetrahydrolipstatin: A derivative of the naturally occurring lipase inhibitor lipstatin. Biochimica et Biophysica Acta, 962(3), 308–316. Buchholz, T., & Melzig, M. F. (2016). Medicinal plants traditionally used for treatment of obesity and diabetes mellitus – Screening for pancreatic lipase and α-amylase inhibition. Phytotherapy Research, 30(2), 260–266. Chen, J., He, X., & Huang, J. (2014). Diet effects in gut microbiome and obesity. Journal of Food Science, 79(4), R442–R451. Conforti, F., Perri, V., Menichini, F., Marrelli, M., Uzunov, D., Statti, G. A., & Menichini, F. (2012). Wild Mediterranean dietary plants as inhibitors of pancreatic lipase. Phytotherapy Research, 26(4), 600–604. de la Garza, A. L., Milagro, F. I., Boque, N., Campión, J., & Martínez, J. A. (2011). Natural inhibitors of pancreatic lipase as new players in obesity treatment. Planta Medica, 77(8), 773–785. Gänzle, M. G. (2015). Lactic metabolism revisited: Metabolism of lactic acid bacteria in food fermentations and food spoilage. Current Opinion in Food Science, 2, 106–117. Giacometti, J., & Buretić-Tomljanović, A. (2017). Peptidomics as a tool for characterizing bioactive milk peptides. Food Chemistry, 230, 91–98. Humbert, G., Guingamp, M. F., Linden, G., & Gaillard, J. L. (2006). The Clarifying Reagent, or how to make the analysis of milk and dairy products easier. Journal of Dairy Research, 73(4), 464–471. Jafar, S., Kamal, H., Mudgil, P., Hassan, H. M., & Maqsood, S. (2018). Camel whey protein hydrolysates displayed enhanced cholesteryl esterase and lipase inhibitory, anti-hypertensive and anti-haemolytic properties. LWT – Food Science and Technology, 98, 212–218. Kang, J. G., & Park, C. Y. (2012). Anti-obesity drugs: A review about their effects and
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safety. Diabetes & Metabolism Journal, 36(1), 13–25. Khanal, S. N., & Lucey, J. A. (2018). Effect of fermentation temperature on the properties of exopolysaccharides and the acid gelation behavior for milk fermented by Streptococcus thermophilus strains DGCC7785 and St-143. Journal of Dairy Science, (18), 30172–30173 pii S0022–0302. Kim, D. H., Kim, H., Jeong, D., Kang, I. B., Chon, J. W., Kim, H. S., Song, K. Y., & Seo, K. H. (2017). Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: Targeted and untargeted community analysis with correlation of biomarkers. The Journal of Nutritional Biochemistry, 44, 35–43. Lim, J., Kale, M., Kim, D. H., Kim, H. S., Chon, J. W., Seo, K. H., Lee, H. G., Yokoyama, W., & Kim, H. (2017). Antiobesity effect of exopolysaccharides isolated from kefir grains. Journal of Agricultural and Food Chemistry, 65(46), 10011–10019. Marrelli, M., Loizzo, M. R., Nicoletti, M., Menichini, F., & Conforti, F. (2014). In vitro investigation of the potential health benefits of wild Mediterranean dietary plants as anti-obesity agents with α-amylase and pancreatic lipase inhibitory activities. Journal of the Science of Food and Agriculture, 94(11), 2217–2224. Moon, Y. J., Soh, J. R., Yu, J. J., Sohn, H. S., Cha, Y. S., & Oh, S. H. (2012). Intracellular lipid accumulation inhibitory effect of Weissella koreensis OK1-6 isolated from Kimchi on differentiating adipocyte. Journal of Applied Microbiology, 113(3), 652–658. Ogawa, A., Kobayashi, T., Sakai, F., Kadooka, Y., & Kawasaki, Y. (2015). Lactobacillus gasseri SBT2055 suppresses fatty acid release through enlargement of fat emulsion size in vitro and promotes fecal fat excretion in healthy Japanese subjects. Lipids in Health and Disease, 14, 20. Park, D. Y., Ahn, Y. T., Huh, C. S., Jeon, S. M., & Choi, M. S. (2011). The inhibitory effect of Lactobacillus plantarum KY1032 cell extract on the adipogenesis of 3T3-L1 Cells. Journal of Medicinal Food, 14(6), 670–675. Park, S. Y., Cho, S. A., Kim, S. H., & Lim, S. D. (2014). Physiological Characteristics and Anti-obesity Effect of Lactobacillus plantarum Q180 Isolated from Feces. Korean Journal for Food Science of Animal Resources, 34(5), 647–655. Park, S. Y., Seong, K. S., & Lim, S. D. (2016). Anti-obesity effect of yogurt fermented by Lactobacillus plantarum Q180 in diet-induced obese rats. Korean Journal for Food
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Lipase inhibitory activity of skim milk fermented with different strains of lactic acid bacteria Ana María Gil-Rodríguez, Thomas P. Beresford
T
⁎
Teagasc Food Research Centre, Moorepark and Food for Health Ireland, Fermoy, Co. Cork P61C991, Ireland
A R T I C LE I N FO
A B S T R A C T
Keywords: Obesity Lactic acid bacteria Lipase inhibitory activity Fermented milk
One of the main causes of obesity is an imbalance between energy intake and expenditure. One approach to facilitate weight loss is to decrease absorption of energy-rich nutrients by inhibition of pancreatic lipase. A spectrophotometric method was developed to evaluate the potential of milk fermented with lactic acid bacteria to inhibit pancreatic lipase in vitro. Fermentates produced with Lactobacillus helveticus strains at 42 °C exhibited the highest levels of lipase inhibition, with strains SC8, SC44 and SC45 inhibiting pancreatic lipase by > 49%, significantly higher than the activity of a milk control. In all cases activity was retained after removal of large proteins including casein and in the < 3 kDa fraction, but not in the 3–10 kDa fraction. Although these results require validation with in vivo models, the strains L. helveticus SC8, SC44 and SC45 are good candidates for use in the production of functional food products with potential anti-obesity effects.
1. Introduction Obesity is an increasing health problem with rising incidence rates, not only in industrialised countries, but also in developing countries. It is usually associated with other pathologic conditions, such as type 2 diabetes mellitus, hypertension, obstructive sleep apnoea and cardiovascular disease (Chen, He, & Huang, 2014; Marrelli, Loizzo, Nicoletti, Menichini, & Conforti, 2014). Apart from appetite suppression, one of the most important therapeutic strategies in the treatment of obesity is the administration of digestion and absorption inhibitors in order to decrease energy intake from the diet. As two major energy sources, the reduction in the absorption of triglycerides and glucose are usually attempted (Marrelli et al., 2014). The main source of dietary fat is triacylglycerol, but this molecule cannot be absorbed in the gut without being hydrolysed into smaller molecules such as monoglycerides and free fatty acids (Tucci, Boyland, & Halford, 2010). Pancreatic lipase is the enzyme responsible for this hydrolysis, subsequently facilitating the absorption of dietary fat. As excessive ingestion of fat is directly related with overweight and obesity, inhibition of this enzyme is a relevant approach for weight loss in addition to reduced-fat diets (de la Garza, Milagro, Boque, Campión, & Martínez, 2011). However, there is only one pharmacologic treatment (Orlistat) currently available to inhibit this enzyme and consequently other inhibitors are being sought (Kang & Park, 2012; Marrelli et al.,
2014; Tucci et al., 2010). Orlistat (tetrahydrolipstatin) is a lipase inhibitor derived from lipstatin, a natural product obtained from the bacteria Streptomyces toxytricini (Borgström, 1988). It inhibits the absorption of fat from the diet by 30%; thus, facilitating weight loss (Kang & Park, 2012). As a result of side effects caused by orlistat (e.g. abdominal pain, diarrhoea, oily stools, liposoluble vitamin deficiencies) and other digestive enzyme inhibitors, new sources are currently being screened in search for milder inhibitors without side effects (Buchholz & Melzig, 2016; Tucci et al., 2010). In this context, the search for lipase inhibitors has focused mostly on herbs and edible plants, but also fungi and bacteria from diverse origins (Reviewed in de la Garza et al., 2011). Apart from reduced side effects, lipase inhibitors naturally present in food products could have the advantage of reducing body weight in obese subjects without the administration of drugs or dietary supplements. Fermented foods (especially fermented dairy products) have traditionally been associated with multiple health benefits. The anti-obesity effect of kefir (Kim et al., 2017; Lim et al., 2017) and milk fermented or supplemented with lactobacilli (Ogawa, Kobayashi, Sakai, Kadooka, & Kawasaki, 2015; Park, Seong, & Lim, 2016; Pothuraju et al., 2016; Sato et al., 2008) has been demonstrated in overweight mice, rats and humans. These (and other) health benefits of fermented dairy products have been linked to milk-derived bioactive peptides (Aguilar-Toalá et al., 2017), which can be released from the native protein through
Abbreviations: NFSM, non-fermented skim milk; RSM, reconstituted skim milk; LAB, lactic acid bacteria; NPC, 4-nitrophenyl octanoate; DMSO, dimethyl sulfoxide ⁎ Corresponding author. E-mail addresses: [email protected] (A.M. Gil-Rodríguez), [email protected] (T.P. Beresford). https://doi.org/10.1016/j.jff.2019.06.015 Received 5 June 2018; Received in revised form 16 May 2019; Accepted 10 June 2019 Available online 16 July 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
2.3. Reconstituted skim milk (RSM) preparation
different mechanisms, one of them being the action of microbial proteolytic enzymes (Qian et al., 2011). In addition, the ability of different strains of lactic acid bacteria (mainly lactobacilli) and bifidobacteria to inhibit pancreatic lipase has been demonstrated in several studies using reconstituted freeze-dried cells (An et al., 2011; Ogawa et al., 2015; Park, Cho, Kim, & Lim, 2014; Zhou et al., 2013). Similarly, some strains of Lactobacillus curvatus, Lactobacillus plantarum and Weisella koreensis have been shown to reduce lipid accumulation and adipogenesis in preadipocyte 3T3-L1 cells (Moon et al., 2012; Park, Ahn, Huh, Jeon, & Choi, 2011). In this study we investigated the potential of milk fermented with different strains of lactic acid bacteria (LAB) from the genera Lactobacillus, Lactococcus and Pediococcus to inhibit pancreatic lipase activity in vitro. In addition, as fermentation temperature has an impact on metabolic activity (including proteolysis) and other parameters such as growth rate of LAB (Khanal & Lucey, 2018; Savijoki, Ingmer, & Varmanen, 2006), we investigated the effect of fermentation temperature in the development of this beneficial activity. Furthermore, we analysed the water soluble fraction following centrifugation of the samples with highest activities and their low molecular size fractions (< 10 kDa, 3–10 kDa and < 3 kDa) in order to determine the molecular range size of the active compounds.
Skim milk powder was reconstituted at 10% (w/v) by slowly adding the powder into distilled water with stirring. Once all the powder was incorporated the milk was left to rehydrate for 2 h with constant stirring. The RSM was sterilised by heat treatment (121 °C, 5 min) to inactivate indigenous microbes, followed by cooling and overnight storage at 4 °C. The milk was then distributed into the fermentation containers and incubated for 1 h at the appropriate fermentation temperature before inoculation with the strain of interest. 2.4. Milk fermentation Fresh overnight cultures (24 h) as described above were used to inoculate (1% v/v) heat-treated 10% RSM. Fermentations were performed at four different temperatures (30, 33, 37 and 42 °C) for each microbial strain and were run for 24 h under aerobic conditions without agitation. During milk fermentation by most LAB lactose is metabolised with production of lactic acid as the main by-product (Gänzle, 2015). Therefore; the pH of the product was measured at the end of the fermentation as an indicator of strain performance in milk. 2.5. Sample fractionation
2. Materials and methods
The samples with the highest lipase-inhibitory activities were subject to fractionation by molecular size in order to isolate the fractions potentially containing bioactive peptides (< 10 kDa) [Aguilar-Toalá et al., 2017, Giacometti & Buretić-Tomljanović, 2017]. The samples were centrifuged (4000 rpm, 30 min) to remove large proteins including casein and the supernatants were sterilised by filtering through a 0.2 µm pore size Polyethersulfone (PES) filters. An aliquot of the resulting filtrate was stored at −20 °C for analysis, and the rest was ultrafiltered in succession using centrifugal filters with different molecular weight cut-offs. First they were pre-filtered through 50 and 30 kDa molecular weight cut-off centrifugal filters in order to remove the molecules with highest molecular weights. The 30 kDa permeate was then filtered through 10 and 3 kDa molecular weight cut-off filters and the fractions < 10 kDa, 3–10 kDa and < 3 kDa were filter-sterilised using a 0.2 µm pore size PES filters and stored at −20 °C for analysis. A control of non-fermented skim milk (NFSM) was also included for reference. In order to separate the supernatant from the control milk the casein was precipitated by acidifying the milk using 0.1 N HCl until a pH of 4.6 was reached (Aguilar-Toalá et al., 2017). After acidification, the control milk was centrifuged under the same conditions as the fermented milk samples and the supernatant was recovered and treated as outlined above.
2.1. Chemicals The anaerobic atmosphere generator Anaerocult A was purchased from OCON Chemicals (Cork, Ireland). De Man, Rogosa, Sharpe (MRS) and M17 culture media were obtained from BD (Oxford, UK). Amicon centrifugal filtration units for sample fractionation were purchased from Merck Millipore (Cork, Ireland). All other reagents used in this study were purchased from Sigma Aldrich (Arklow, Ireland).
2.2. Microbial strains and culture conditions In this study, 31 strains of LAB belonging to the DPC culture collection (Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland) were used. These strains were selected among 7 different species of the genera Lactobacillus, Lactococcus and Pediococcus (Table 1) with QPS (qualified presumption of safety) status. The strains of Lactobacillus and Pediococcus were grown in MRS supplemented with 0.5 g/L L-cystine hydrochloride and incubated at 37 °C under anaerobic conditions generated using Anaerocult A, whereas Lactococcus strains were grown at 30 °C in M17 supplemented with 10 g/L α-lactose. Before milk fermentation each strain was transferred from a frozen glycerol stock culture maintained at −20 °C onto an agar plate and incubated 48 h. A single colony was isolated onto a fresh agar plate to ensure culture purity. From this second plate a liquid culture was inoculated. This fresh overnight culture (24 h) was in turn used to seed (1% v/v) a second broth that would be used to inoculate the milk.
2.6. Peptidic profile The peptidic profile of the samples with highest lipase inhibitory activity, their supernatants and the active fractions (< 3 kDa) were obtained as described by Slattery and Fitzgerald (1998). Briefly, 200 µL of each sample were diluted in 9.8 mL of distilled water and filtered through a 0.2 µm Polyvinylidene Fluoride (PVDF) filter. The dilutions were subsequently analysed by size exclusion chromatography (SEC) using a TSK G2000SW (300 × 7.5 mm) and a TSK G2000swxl column (300 × 7.8 mm, Tosu Hass, Japan) in series, fitted to a Waters Alliance 2695 separation module (Waters Corporation, Milford, Mass, USA). The mobile phase used was prepared by adding HPLC grade acetonitrile (30% v/v) and trifluoroacetic acid (0.1% v/v) to R2 filtered water. Assays were run using a flowrate of 1 mL/min and continually monitored at 214 nm using a Waters 2487 dual wavelength detector. Chromatographic data were collected and analysed using Empower data handling software package (Waters Corporation, Milford, MA, USA). Standard proteins and peptides were used to prepare a retention time to molecular weight calibration curve.
Table 1 Species of LAB and number of strains per species used in this study. Species
No. strains
Lactobacillus acidophilus Lactobacillus brevis Lactobacillus delbrueckii ssp. bulgaricus Lactobacillus helveticus Lactobacillus plantarum Lactococcus lactis Pediococcus pentosaceus
4 2 2 13 4 3 3
2
Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
Table 2 Milk pH values after 24 h fermentation at different temperatures. Species
Strain no.
pH 30 °C
L. acidophilus
L. brevis L. delbrueckii ssp bulgaricus L. helveticus
L. plantarum
Lc. lactis
P. pentosaceus
SC1 SC2 SC3 SC63 SC37 SC92 SC40 SC77 SC4 SC5 SC6 SC7 SC8 SC41 SC42 SC43 SC44 SC45 SC46 SC47 SC64 SC70 SC71 SC72 SC80 SC15 SC18 SC34 SC29 SC30 SC57
6.11 5.88 5.88 5.76 6.39 6.43 4.40 4.25 3.95 5.13 5.00 4.09 4.86 4.75 4.79 4.84 5.01 4.82 5.72 4.64 5.46 4.89 4.94 4.92 5.20 4.28 4.25 4.26 6.04 6.04 6.12
33 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.09 0.03 0.09 0.00 0.01 0.20 0.13 0.07 0.03 0.12 0.01 0.08 0.30 0.14 0.04 0.05 0.10 0.11 0.05 0.08 0.04 0.06 0.04 0.06 0.01 0.00 0.01 0.12 0.06 0.00
5.54 5.22 5.15 5.24 6.39 6.34 3.76 3.72 3.66 4.56 4.01 3.72 3.97 3.97 3.93 3.96 3.96 3.88 5.37 3.72 5.00 4.75 4.75 4.75 5.12 4.38 4.33 4.31 5.93 5.89 6.09
37 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.00 0.04 0.04 0.06 0.00 0.16 0.12 0.03 0.04 0.11 0.03 0.13 0.21 0.06 0.12 0.09 0.00 0.12 0.03 0.04 0.08 0.06 0.07 0.08 0.04 0.04 0.03 0.16 0.07 0.05
5.47 4.83 4.87 4.83 6.49 6.47 3.92 3.74 3.57 3.95 3.69 3.68 3.73 3.59 3.61 3.60 3.60 3.60 5.24 3.58 4.60 4.67 4.70 4.70 5.09 4.49 4.44 4.43 5.94 5.87 6.03
42 °C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.43 0.47 0.04 0.07 0.06 0.17 0.12 0.08 0.01 0.17 0.18 0.23 0.13 0.10 0.10 0.12 0.10 0.02 0.05 0.04 0.04 0.07 0.06 0.12 0.00 0.05 0.04 0.16 0.07 0.01
4.79 5.00 4.91 4.87 6.52 6.53 3.75 3.72 3.59 3.84 3.61 3.53 3.46 3.48 3.53 3.65 3.42 3.43 5.04 3.41 4.21 5.58 5.62 5.61 5.79 6.25 5.03 5.01 5.98 6.37 6.07
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.04 0.10 0.04 0.06 0.10 0.08 0.05 0.21 0.19 0.11 0.26 0.11 0.09 0.07 0.10 0.21 0.02 0.02 0.10 0.02 0.16 0.31 0.42 0.44 0.05 0.19 0.16 0.20 0.02 0.04 0.09
measurements. Apart from eliminating the turbidity due to the addition of milk to the reaction mixture, the clarification step also stops the enzymatic reaction between lipase and NPC due to a pH change (Humbert, Guingamp, Linden, & Gaillard, 2006). The mixture was gently mixed and subsequently incubated 3 min at 37 °C. After incubation, absorbance was measured at 412 nm using a Jenway 6300 Spectrophotometer (Cole-Parmer Ltd, Staffordshire, UK). Due to the increase in the buffer concentration, turbidity still remained in some of the samples. These samples were filtered using a 0.2 µm PES filter before absorbance measurement. The percentage of lipase inhibitory activity was calculated with respect to 100% activity reference control as follows:
2.7. Lipase inhibitory activity test For the detection of lipase inhibitory activity in the milk fermentates, a method was developed by adapting the protocols described by Bendicho, Trigueros, Hernández, and Martin (2001), Conforti et al. (2012) and Marrelli et al. (2014) with modifications. The pH of the reaction mixture is critical for the hydrolysis of 4nitrophenyl octanoate (NPC) into octanoate and 4-nitrophenol (yellow coloured product) by pancreatic lipase to proceed. The concentration of the buffers described in the cited literature (Tris-HCl between 0.05 and 0.07 M) were not sufficient to maintain the pH of the reaction at 8.5 after addition of the most acidic milk samples (pH ≤ 4.5). In order to maintain the pH in the optimal range for lipase activity under these conditions (8.25–8.75), increasing concentrations of Tris-HCl were tested. It was observed that at concentrations of Tris-HCl ≥ 0.15 M, the clarifying reagent for dairy products was unable to eliminate the turbidity of the samples (data not shown) and thus, a two buffer system was developed. For samples with pH > 4.5 and the reference control, Tris-HCl 0.10 M at pH 8.5 was used, whereas for samples with pH ≤ 4.5, Tris-HCl 0.10 M buffer was prepared at pH 9.5. To test the lipase inhibitory activity of the different fermented milks, 500 µL of sample were mixed with 2 mL of Tris-HCl buffer, following which 50 µL of a 5 mM solution of NPC in dimethyl sulfoxide (DMSO) were added. The reaction was initiated by adding 50 µL of a 5 mg/mL solution of pancreatic lipase (Type II, from porcine pancreas) dissolved in Tris-HCl buffer pH 8.5. The mixture was vortexed for 2 min at medium speed to avoid the formation of foam and incubated at 37 °C for 30 min. For the reference control, 500 µL of Tris-HCl buffer were added instead of sample. A blank was prepared for each sample, where NPC was substituted by 50 µL DMSO. A control using orlistat in DMSO (10 µg/mL) was included for comparison. After 30 min incubation at 37 °C, 1 mL of Clarifying Reagent for Dairy Products was added to each tube to allow absorbance
AbsS − AbsSB × 100⎞ Lipase inhibitory activity (%) = 100 − ⎛ ⎝ AbsRC − AbsB ⎠ ⎜
⎟
where AbsS is the absorbance of the sample, AbsSB is the absorbance of the corresponding blank, AbsRC is the absorbance of the reference control and AbsB is the absorbance of the blank of the reference control. The presence of milk in the reaction mixture provokes a reduction in the absorbance of p-nitrophenol of 5.71%. This value was experimentally obtained (data not shown) and applied as a correction factor to the net absorbance values before calculating the percentages of inhibition.
2.8. Statistical analysis Each milk fermentate was produced three times and analysed twice in duplicates. The results are expressed as average ± standard deviation. Significant differences were analysed with Pearson's chi-squared test. Values of P < 0.01 were considered statistically significant.
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3. Results
However, for strains of other species the opposite trend is observed, e.g., two out of the three Lc. lactis strains tested (SC18 and SC34) exhibited levels of lipase inhibitory activity significantly different from the control (35.44 ± 2.62% for SC18 and 38.29 ± 2.26% for SC34) when the fermentation was performed at 42 °C, but not when performed at lower temperatures (Fig. 1d). Contrarily as to what was observed for L. helveticus strains, for Lc. lactis SC18 and SC34 the lowest pH is reached when fermentation takes place at 30 °C (4.25 ± 0.00 for SC18 and 4.26 ± 0.01 for SC34) and increases progressively until it reaches its maximum at 42 °C (5.03 ± 0.16 for SC18 and 5.01 ± 0.20 for SC34), thus indicating a decreasing production of lactic acid as temperature increases, as would be expected (the optimum growth temperature for Lc. lactis is ∼30 °C). It is noteworthy that for this species, the maximum lipase inhibitory activity seems to coincide with the lower performance in milk and decreases together with pH. The strain Lc. lactis SC15 shows a trend similar to SC18 and SC34, but none of the activities registered for this strain are significantly different from the control. The other strains analysed, belonging to species L. acidophilus, L. brevis, L. delbrueckii ssp bulgaricus, L. plantarum and P. pentosaceus show moderate maximum activities with values around 35% inhibition (Fig. 1). L. acidophilus SC1 and SC63 show a similar profile, with higher activities at 37 and 42 °C, whereas SC2 and SC3 have their maximum at 33 °C. The two strains of L. brevis and the three strains of P. pentosaceus tested show no notable differences in pH at different fermentation temperatures, but some of the strains exhibit peaks of moderate activity at specific temperatures (e.g., 33 °C for P. pentosaceus SC29 and 37 °C for L. brevis SC37 and P. pentosaceus SC30). All strains of L. plantarum, except SC80, display a similar tendency to L. helveticus SC46 and SC64 with sequential increases in lipase inhibitory activity with rising fermentation temperatures until a maximum activity is reached at 37 °C. The strains L. plantarum SC70, SC71 and 72 show very similar profiles, although the activities obtained for strain SC70 are higher (maximum activity 36.83 ± 3.77% at 37 °C) than those obtained for SC71 and SC72. Interestingly, in all L. plantarum strains the pH values of the fermented milks decrease sequentially between 30 and 37 °C, but increase again at 42 °C, where lipase inhibitory activity decreases. Contrarily to what was observed for other strains of this species, inhibition levels by strain L. plantarum SC80 do not vary with temperature. Apart from SC80, this apparent lack of influence of fermentation temperature on lipase inhibitory activity is observed only with P. pentosaceus SC57. Although some strains from the same species show similar levels of lipase inhibition in their milk fermented products at a given temperature, the potential to inhibit pancreatic lipase seems strain-dependent (e.g., L. helveticus, Lc. lactis). Also, our data suggest that changes in the metabolic activity of LAB provoked by differences in fermentation temperature can strongly influence the development of lipase inhibitory activity (and potentially other bioactivities) during milk fermentation.
3.1. Strain performance in milk The reduction in pH with respect to NFSM (6.47 ± 0.03) was measured as an indicator of strain performance. Most of the strains used in this study reduced the pH of milk to some extent after 24 h fermentation at different temperatures, with Lactobacillus brevis strains as only exception (pH values between 6.34 and 6.53). Similarly, the pH reduction observed in milk fermented with Pediococcus pentosaceus was low, with values between 5.87 and 6.12 (Table 2). Generally, strains of the same species resulted in similar pH values after fermentation at a given temperature. However, some strains e.g., Lactobacillus helveticus SC46 and SC64 and L. plantarum SC80 at all temperatures and Lactococcus lactis SC15 at 42 °C all resulted in lower pH reductions to other strains of the same species under identical fermentation conditions. The lowest pH values (< 4) were registered for L. helveticus strains at 42 °C, increasing progressively as fermentation temperature decreased but staying below 4 at 37 and 33 °C for most strains. These were closely followed by Lactobacillus delbrueckii ssp bulgaricus strains with pH values below 4 for fermentates produced at 42, 37 and 33 °C. The other strains used in this study provoked more moderate decreases in pH. Lc. lactis strains acidified milk to values between 4.25 and 4.5 at 30, 33 and 37 °C, with the lowest values at 30 °C increasing progressively with temperature. Contrarily, milk fermented with Lactobacillus acidophilus and L. plantarum strains had their minimum pH value at 37 °C, increasing progressively as fermentation temperature decreased. 3.2. Development of lipase inhibitory activity during milk fermentation Under these experimental conditions (see Section 2.7 of Materials and methods), 10 µg/mL orlistat inhibited pancreatic lipase by 70.17 ± 4.61%. The highest levels of lipase inhibitory activity reported in this study were obtained for milk fermented with different strains of L. helveticus at 42 °C, with 8 products displaying inhibition levels above 45% (SC6, SC7, SC8, SC41, SC42, SC43, SC44 and SC45). Of these products, the highest activities were registered for strains SC8 (49.30 ± 3.56%), SC44 (49.43 ± 2.39%) and SC45 (49.75 ± 2.13%) (Fig. 1a). Overall, most of the milks fermented with L. helveticus strains developed high levels of lipase inhibitory activity at all tested temperatures, with some exceptions at 30 and 33 °C. The only milks whose levels of lipase inhibition did not reach 35% in any of the test conditions were those fermented with SC5, SC46 and SC64. In two of these (SC46 and SC64) activity increased with fermentation temperature until moderate levels were reached at 37 °C, but the activity decreased when fermentation temperature was further increased to 42 °C. Interestingly, the pH of the milk fermented by these two strains decreased with increasing temperature, having its highest value at 30 °C and the lowest at 42 °C (Table 2). As lactose fermentation leads to the formation of lactic acid and a subsequent decrease in pH, a lower pH value can be indicative of a higher level of metabolic activity and/or microbial growth. However, the pH values that these two strains attain after fermentation at 42 °C (5.04 ± 0.10 for SC46 and 4.21 ± 0.16 for SC64) are markedly different from the pH that the other strains of this species attained at this temperature (between 3.41 and 3.84). This suggests that these strains are metabolically different from the other strains analysed, thus possibly explaining the differences in lipase inhibitory activity observed between these two strains and the others from the same species. This could indicate that, for this species, the maximum activity of each strain is usually obtained in the product whose pH is lower, thus suggesting that metabolic activity may have an influence on the development of lipase inhibitory activity. This tendency can also be observed in strains of other species, such as L. delbrueckii ssp bulgaricus SC40 and SC77 (Fig. 1b) and L. acidophilus SC63 (Fig. 1c).
3.3. Analysis of water soluble fractions The three samples with highest lipase inhibitory activity (i.e., milk fermented by L. helveticus SC8, SC44 and SC45 at 42 °C) were selected for fractionation as described in Materials and methods. In summary large proteins (mostly caseins) were removed by centrifugation and the supernatants were filtered through a range of molecular weight cut-off filters resulting in fractions < 10 kDa, 3–10 kDa and < 3 kDa. As shown in Fig. 2, lipase inhibitory activity is retained in the supernatant from all three samples. All the samples analysed showed a markedly higher activity than the supernatant from NFSM with statistically significant differences. A notable reduction in the background activity of NFSM can be observed when casein is removed, as demonstrated in the reduction from 24.43 ± 1.20% to 2.54 ± 1.57% recorded in the NFSM samples for fermented milk and supernatant respectively. A reduction in lipase inhibitory activity was also noted for 4
Journal of Functional Foods 60 (2019) 103413
A.M. Gil-Rodríguez and T.P. Beresford
Fig. 1. Lipase inhibitory activity of milk fermented with different species of LAB at different temperatures (30, 33, 37 and 42 °C). The value obtained for the NFSM control is indicated as a horizontal bar, * indicates a significant difference between that sample and the NFSM control.
Interestingly, the activity observed after analysing the < 10 kDa fraction of all samples can be observed in the < 3 kDa fraction but not in the 3–10 kDa fractions, thus indicating that the activity observed is produced by a molecule with molecular size < 3 kDa.
the three supernatants relative to their respective fermented milk, further supporting the suggestion that the background activity in NFSM is mostly due to the presence of casein. The < 10 kDa fractions of all three samples exhibit levels of lipase inhibitory activity comparable to the levels observed in their respective supernatants before fractionation, albeit slightly reduced. This reduction; however, is only statistically significant in the sample SC8. As bigger proteins and molecules are removed during the ultrafiltration process, these results indicate that the inhibition of pancreatic lipase observed in these samples is produced by a medium or small sized molecule (below 10 kDa molecular weight), possibly a peptide released by hydrolysis of milk proteins by microbial proteolytic enzymes.
3.4. Peptidic profiles Peptidic profiles were obtained by size exclusion chromatography (SEC) as described in the materials and methods section and are presented in Fig. 3. It can be observed that the profiles of the three analysed fermentates and their associated supernatants and < 3 kDa fractions are very similar, suggesting similar proteolytic activities in the Fig. 2. Lipase inhibitory activity of the fermentates with the highest activities (L. helveticus SC8, SC44 and SC45 at 42 °C) and their respective fractions. a above the bar indicates a significant difference between that sample and the NFSM control. For comparison between the supernatants and small molecular size fractions (< 10 kDa, 3–10 kDa and < 3 kDa) of each fermentate, b indicates a statistically significant difference between that fraction and its respective supernatant prior to fractionation.
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Fig. 3. Peptidic profiles of the fermentates with the highest activities (L. helveticus SC8, SC44 and SC45 at 42 °C) and their respective supernatants and < 3 kDa fractions: (a) SC8, (b) SC44, (c) SC45 and (d) NFSM control. The retention times highlighted in the profiles are, from left to right 12.181 min (molecules > 20 kDa), 14.979 min (20–10 kDa), 15.000 min (10–5 kDa), 18.000 min (5–2 kDa), 19.169 min (2–1 kDa), 20.226 min (1–0.5 kDa) and 24.596 min (< 0.5 kDa).
mw < 2 kDa (Fig. 3a–c). This suggests that milk proteins are completely hydrolysed by these strains into smaller peptides. As corresponds to their low molecular size, these two peaks can also be observed in the < 3 kDa fraction, which exhibited high lipase inhibitory activity in all cases. This suggests that either (a) one of the two peaks represents the active component or (b) both components are necessary to inhibit pancreatic lipase.
three strains used to produce these samples. In two of the NFSM profiles (intact sample and supernatant), two peaks can be observed (Fig. 3d), one in the area corresponding to molecules of mw > 20 kDa, which could correspond to caseins (23 kDa for β-casein and 24–26 kDa for α-casein), and a smaller peak in the area of 10–20 kDa, which could correspond to β-lactoglobulin, α-lactalbumin (18 kDa and 14 kDa, respectively), and κ-casein (19 kDa) (Vincent et al., 2016). In contrast, all the profiles of fermented milk samples (SC8, SC44 and SC45) and their fractions do not exhibit these peaks, but present two peaks in the area corresponding to molecules of
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4. Discussion
effect on overweight, obesity and related conditions. However, the mechanisms whereby these beneficial effects are achieved need to be further elucidated.
The anti-obesity effect of different species of LAB (mainly lactobacilli) and bifidobacteria has been reported in many studies, but only a few have investigated the potential of these beneficial microorganisms to inhibit pancreatic lipase in vitro (Ogawa et al., 2015; Park et al., 2014; Zhou et al., 2013) and, in all cases, the studies were performed on resuspended freeze-dried bacterial cells from washed cultures. In a similar study, peptidic fractions obtained by enzymatic hydrolysis of camel milk whey were reported to exhibit pancreatic lipase inhibitory activity levels between 25 and 55% (Jafar, Kamal, Mudgil, Hassan, & Maqsood, 2018). To our knowledge, the current study is the first where the potential anti-obesity effect of milk fermented with different strains of LAB and their derived low molecular size fractions has been assessed in vitro. The activities reported in the present study are moderate when compared to orlistat at 10 µg/mL (70.17 ± 4.61%). In line with these results, Park et al. (2014) reported that L. plantarum Q180 inhibits pancreatic lipase by 83.61 ± 2.31% at concentrations of 100 µg/mL by directly inhibiting the hydrolysis of p-nitrophenylpalmitate. Comparison of inhibition levels between the two studies is difficult; however, as the methods for lipase inhibition assessment are notably different despite being based on the release of a coloured product resulting from enzymatic hydrolysis. Furthermore, the activity is measured in different types of sample (resuspended lyophilised bacterial cells vs. fermented milk and water soluble fractions after casein removal). The mechanism whereby lipase inhibition is achieved in these fermented milk products is unclear and several mechanisms may be involved. In a recent study, the capacity of Lactobacillus gasseri LG2055 to inhibit pancreatic lipase in vitro was tested with two different methods: i) hydrolysis of 4-methylumbelliferyl oleate and ii) decrease in fat droplet size. In contrast to our results, the data obtained by these authors indicate that the activity of this strain is not due to a direct inhibition of the enzyme (no decrease in the hydrolysis of 4-methylumbelliferyl oleate is observed) but due to an increase in fat droplet size. This results in a decrease in the oil-water interface surface, which subsequently hinders lipase activity. These findings were related to a significant increase in faecal fat excretion in healthy adults after administration of milk fermented with this strain (Ogawa et al., 2015). Zhou et al. (2013) demonstrated that Lactobacillus pentosus Z-PT84 inhibited the hydrolysis of high-oleic safflower oil by pancreatic lipase. The authors hypothesised that the hydrophobicity of the cell surface may facilitate adhesion of the bacterial cell to the oil-water interface (where pancreatic lipase exerts its activity) and modify its mechanical properties, thus affecting lipase activity. In this context; the more hydrophobic the cell surface, the higher the potential for adhesion to the substrate interface and therefore, the higher the interference with lipase activity. In the present study the activity of the selected samples is retained after removal of casein and microbial cells during the filtration and fractionation process. Furthermore, the activity is retained in the < 3 kDa fraction in all three samples tested, which contained two groups of low molecular weight peptides (< 2 kDa), as demonstrated in the two peaks observed in the SEC profiles of all three samples. This suggests that in this case lipase is inhibited by a small size milk-derived peptide released during microbial fermentation. In line with our results, other studies have reported diverse bioactivities in the < 3 kDa fractions of milk fermented with different strains of LAB, and generally the activity of these fractions was higher or at least equal to the activity of the 3–10 kDa fractions (Aguilar-Toalá et al., 2017; Qian et al., 2011). In addition, peptides from milk kefir have recently been shown to modulate lipid metabolism in vivo by regulating the genetic expression of different enzymes involved in both lipogenesis and lipid oxidation in the liver (Tung et al., 2018). Collectively, these results present strong evidence of the potential of milk-derived bioactive peptides released by microbial fermentation to regulate lipid metabolism at different stages and exert a beneficial
5. Conclusions Our results indicate that different strains of LAB have the ability to produce fermented milks with the capacity to partially inhibit pancreatic lipase in vitro. Of all the products tested, milk fermented with L. helveticus SC8, SC44 and SC45 at 42 °C are good candidates as functional foods for weight loss and improvement of obesity-related pathologic conditions with reduced side effects. However, further studies are needed to evaluate the anti-obesity potential of these milk fermented products in appropriate in vivo models. The lipase inhibition activity associated with these fermented milks were observed in filter sterilised fractions containing low molecular weight molecules (< 3 kDa). Furthermore, these fractions exhibit two peaks corresponding to small size (< 2 kDa) peptides. These data suggest that the active component is a peptide released from a milk protein hydrolysed during fermentation by L. helveticus. The data reported here were generated using an in vitro assay for pancreatic lipase activity suggesting that the mechanism of action relates to inhibition of the enzyme, possibly by inhibition of the its active site or interference with a critical location on the enzyme. In addition, the method described in this paper could be considered as a first-step screening for fermented dairy products with potential anti-obesity activity. Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgements This work was supported by Food for Health Ireland (Enterprise Ireland Grant: CC20080001). References Aguilar-Toalá, J. E., Santiago-López, L., Peres, C. M., Peres, C., Garcia, H. S., VallejoCordoba, B., González-Córdova, A. F., & Hernández-Mendoza, A. (2017). Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains. Journal of Dairy Science, 100(1), 65–75. An, H. M., Park, S. Y., Lee, D. K., Kim, J. R., Cha, M. K., Lee, S. W., Lim, H. T., Kim, K. J., & Ha, N. J. (2011). Antiobesity and lipid-lowering effects of Bifidobacterium spp. in high fat diet-induced obese rats. Lipids in Health and Disease, 10, 116. Bendicho, S., Trigueros, M. C., Hernández, T., & Martin, O. (2001). Validation and comparison of analytical methods based on the release of p-nitrophenol to determine lipase activity in milk. Journal of Dairy Science, 84(7), 1590–1596. Borgström, B. (1988). Mode of action of tetrahydrolipstatin: A derivative of the naturally occurring lipase inhibitor lipstatin. Biochimica et Biophysica Acta, 962(3), 308–316. Buchholz, T., & Melzig, M. F. (2016). Medicinal plants traditionally used for treatment of obesity and diabetes mellitus – Screening for pancreatic lipase and α-amylase inhibition. Phytotherapy Research, 30(2), 260–266. Chen, J., He, X., & Huang, J. (2014). Diet effects in gut microbiome and obesity. Journal of Food Science, 79(4), R442–R451. Conforti, F., Perri, V., Menichini, F., Marrelli, M., Uzunov, D., Statti, G. A., & Menichini, F. (2012). Wild Mediterranean dietary plants as inhibitors of pancreatic lipase. Phytotherapy Research, 26(4), 600–604. de la Garza, A. L., Milagro, F. I., Boque, N., Campión, J., & Martínez, J. A. (2011). Natural inhibitors of pancreatic lipase as new players in obesity treatment. Planta Medica, 77(8), 773–785. Gänzle, M. G. (2015). Lactic metabolism revisited: Metabolism of lactic acid bacteria in food fermentations and food spoilage. Current Opinion in Food Science, 2, 106–117. Giacometti, J., & Buretić-Tomljanović, A. (2017). Peptidomics as a tool for characterizing bioactive milk peptides. Food Chemistry, 230, 91–98. Humbert, G., Guingamp, M. F., Linden, G., & Gaillard, J. L. (2006). The Clarifying Reagent, or how to make the analysis of milk and dairy products easier. Journal of Dairy Research, 73(4), 464–471. Jafar, S., Kamal, H., Mudgil, P., Hassan, H. M., & Maqsood, S. (2018). Camel whey protein hydrolysates displayed enhanced cholesteryl esterase and lipase inhibitory, anti-hypertensive and anti-haemolytic properties. LWT – Food Science and Technology, 98, 212–218. Kang, J. G., & Park, C. Y. (2012). Anti-obesity drugs: A review about their effects and
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