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resistant starch microcapsules controlling nisin release
Journal Pre-proofs Novel design for alginate/resistant starch microcapsules controlling nisin release Hebatoallah Hassan, Ahmed Gomaa, Muriel Subirade, Ehab Kheadr, Daniel St-Gelais, Ismail Fliss PII: DOI: Reference:
S0141-8130(19)35976-8 https://doi.org/10.1016/j.ijbiomac.2019.10.248 BIOMAC 13749
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
30 July 2019 24 October 2019 26 October 2019
Please cite this article as: H. Hassan, A. Gomaa, M. Subirade, E. Kheadr, D. St-Gelais, I. Fliss, Novel design for alginate/resistant starch microcapsules controlling nisin release, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.248
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© 2019 Published by Elsevier B.V.
Novel design for alginate/ resistant starch microcapsules controlling nisin release Hebatoallah Hassan a,b,c, Ahmed Gomaa a,d1, Muriel Subirade a, Ehab Kheadr c, Daniel St-Gelais e & Ismail Fliss a1 a STELA
Dairy Research Center, Food sciences department, Institute of Nutrition and Functional Foods (INAF), Université Laval, Québec, Canada b Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt c Faculty of Agriculture, Functional Foods and Nutraceuticals Lab, Alexandria University, Alexandria, Egypt d Nutrition and Food Science Department, National Research Center, Cairo, Egypt e Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Canada
Abstract This study aimed to develop a novel nontoxic, biocompatible, biodegradable, and cost-efficient matrix for the encapsulation of antimicrobial component (nisin) to be used as bio-preservative agent in cheddar cheese. Nisin A loaded beads were prepared from alginate at 0.5 %, 1% and 2 %; and hi-maize resistant starch at 0.5 or 1 %. Beads were characterized by microscopic examination and transmission electron microscopy. Molecular structures were investigated by FTIR, and particle size distributions were measured. The Entrapment efficiency (EE) was measured microbiologically by agar diffusion. The encapsulated nisin showed similar inhibition activities in all developed formulas with an inhibition zone of 15±2 mm. The FTIR analysis confirmed the compatibility of the nisin with sodium alginate and starch. The formulas composed of 1 % Alginate and 0.5 % Non-Gelatinized Starch had the highest encapsulation efficiency among other formulas (33 %). Moreover, that formula allowed the protection and gradual release of the encapsulated
1
Corresponding author: Institute INAF, Laval University, Pavillon Paul-Comtois, Québec, Canada. Fax : +1 (418) 656-3353; E-mail : [email protected]
nisin during a long-term storage for up to two months. Application in cheddar cheese proved the inhibition of encapsulated nisin on the growth of C. tyrobutyricum at the large-scale production. In conclusion, the alginate (Alg)/ non-gelatinized resistant starch formula is suitable for the protection and controlled release of nisin in food applications. Keywords: Encapsulation; nisin; Cheddar cheese 1. Introduction Recently, there is an increasing interest in applying bio-preservatives in food products instead of chemical preservatives. Bio-preservation or biocontrol refers to the use of natural or controlled microbiota, or its antimicrobial products against food spoilage or pathogenic microbes to extend the shelf life and enhance the safety of foods [1]. According to Davidson et al., 2013 [2], the antimicrobial component should be nontoxic, effective at low concentrations in its natural form, economical to be applicable in commercial scale, also, does not affect the sensory parameters of the food product, in addition it inhibit a wide range of pathogenic and spoilage microorganisms [2]. Microbes usually live in complex ecosystems where they must interact with the surrounding components of the environment. They compete with each other for space and nutrients in order to survive. One of the most important competition strategies is based on the modification of the environment conditions by the release of antimicrobial substances as by-products of their normal metabolic activity that inhibit growth or even kill the competitors. It can be more specific encoded by specific genetic determinants aimed to combat against specific microbes [3]. lactic acid bacteria (LAB) have a great potential for extended use in bio-preservation. LAB exist naturally in many food systems and conceded as the cornerstone for developing fermented food
sector, also have a long history of safe use in fermented foods, and are classified as Generally Regarded as Safe (GRAS). During fermentation, LAB produce various antimicrobial compounds such as organic acids, diacetyl, hydrogen peroxide, bacteriocins or antifungal molecules [3]. Bacteriocins that are produced by lactic acid bacteria have a promising potential application as bio-preservatives. Although a great number of bacteriocins are discovered, nisin is the only one permitted for using in food industry [4]. Nisin is recognized as safe (GRAS) by FAO/WHO and Expert Committee on Food Additives. It has been used as bio-preservative in meat, vegetables and dairy products [5]. Nisin has a wide spectrum of antibacterial activity against gram positive bacteria and heat stable. However, nisin easily interacts with food ingredients like proteins, lipids and carbohydrates, which lead to a rapid inactivation. In addition, it is affected by the presence of proteolytic enzymes, which reduce its activity against food pathogenic or spoilage microorganisms [6, 7]. To overcome these limitations, encapsulation is a process that could be implemented in order to protect nisin from food matrix, provide and maintain the gradual release of nisin during food storage. Several matrices have been used in literatures for the encapsulation of nisin like liposomes (Laridi, Kheadr et al. 2003), alginate, chitosan or a mixture of polymers such as chitosan/ monolaurin [8]; amaranth / pullulan [9];zein capsules [10]; nanoparticles using poly-g-glutamic acid (g-PGA) and poly-Llysine (PLL) [11]; chitosan-coated nisin-silica [12] or polyethylene oxide nanofibers containing nisin-loaded poly-g-glutamic acid/chitosan. [13]. However, some drawbacks were attributed to these materials, including low encapsulation efficiency, instability, high cost and/or not applicable for large scale production. [8, 9, 14, 15]. In this study, we have used alginate and resistant starch to design a novel encapsulation matrix for nisin. Alginate is a biodegradable, food grade polymer, simple structure and easy to form capsules
in the presence of cations in aqueous solution [15, 16]. However, micro-particles produced from alginate alone exhibited physical instability, porous and hydrophilic structure which lead to rapid release of bioactive material [17, 18]. The additions of other materials like polysaccharides (starch, guar gum or pectin) , polysaccharide nano-crystals have been used to improve the stability and release of alginate [15, 19]. In another study, Chakraborty, Khandai, Sharma, Khanam, Patra, Dinda and Sen [18] used microcapsules from sodium alginate and pectin for encapsulation of anti-inflammatory drug to get sustained drug release in phosphate buffer at pH 6.8 for 12 hours compared to the microcapsules composed of sodium alginate alone. Due to the rigid coat that provided by pectin when it formed a gel in the presence of calcium during ion-exchange gelation. Starch is like alginate as a biodegradable polymer, nontoxic, biocompatible to food matrices and available at low cost [20]. Compared to native starch, resistant starch is resistant to enzymatic lysis which presents naturally in food matrices or produced by bacteria in case of fermented food products. Accordingly, resistant starch is stable during food storage and provide controlled release of bioactive material [20, 21]. Several studies used the starch and its derivatives like cross-linked starch,
starch-methacrylic
acid,
starch-ethylene
vinyl
alcohol
and
starch-2-
hydroxyethylmethacrylate as probiotics and peptide delivery system [22-25]. Recently, some studies have tried to encapsulate nisin for food applications using alginate alone with vibrating technology. Although the particle size achieved using vibrating technology are homogenic, the antimicrobial activity of nisin decreased after 168h at 4 ˚C and 72 hours at 20 ˚C [16]. Alginate and resistant starch polymers could form a promising mixture with the ability of largescale production for food application. Moreover, these mixtures are food grade microcapsules at
low cost. In literature there are very few studies that investigated alginate and starch. In this regard, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] used 2% of resistant starch with sodium alginate to encapsulate nisin by emulsion method. They found that, the presence of resistant starch increased the time of release of 50 % of the nisin up to one week longer than the formula composed of alginate alone. However, there is a gap in literature that needs more studies using different concentrations of starch and sodium alginate, in addition to, investigating the release during long period of storage in conditions mimic to food matrices. The aim of this study was to develop novel matrices for the encapsulation of antimicrobial component (nisin) to be used as bio-preservative agent for cheddar cheese. The encapsulation matrix was developed from alginate and resistant starch in order to improve the properties of each polymer and overcome the limitations. According to our knowledge, there are no previous studies about the production of microcapsules using alginate/ resistant starch by ionotropic gelation vibration technology. Also, the effect of the physical state of starch, gelatinized or non-gelatinized, on the structure and function properties of capsules has not been addressed. Moreover, no previous research investigated the effect of different combinations from alginate and starch at different concentrations in the characteristics of microcapsules. Finally, this study is the first study to investigate structure, stability, and storage of microcapsules during long term of storage. 2. Material and methods Resistant starch was obtained from Manitoba Strach products Inc, Canada. All bacterial culture media were purchased from Nutri Bact, Canada. Sodium alginate and calcium chloride were obtained from Sigma Aldrich, Canada. Cheddar cheese starter culture, L. lactis Subsp. cremoris
CUC222 and L. lactis Subsp. lactis CUC-H, were obtained from Agriculture and Agri-Food Canada, St-Hyacinthe, Canada. 2.1. Bacterial preparation Pediococcus acidilactici UL5 Strain obtained from Laval university culture collection. It was used as indicator strains in the nisin activity assay. Bacterial strain activated twice for 16 h at 37 ºC of incubation in MRS media before using. Spores production Clostridium tyrobutyricum ATTC 25755 was used. The strain was activated twice in Reinforced Clostridial Medium (RCM). Spores were prepared according to [27] with some modifications. The strain was inoculated at 1 % in RCM media and incubated two weeks at 37 ºC in anaerobic chamber. The spores were collected by centrifugation at 5000 g at 4 ºC for 10 min. the spores were resuspended in skim milk media (12%). The spores were enumerated using RCM media after heating at 85 ºC for 5 min to activate the germination of spores. 2.2.Nisin preparation Commercial nisin was obtained from Cayman chemical, USA. Commercial nisin was purified by Sep-Pack C18 column at 4 °C, at a flow rate of 3 ml/min. Followed by a concentration step by Speed-Vac for 6h at 50 °C. The nisin concentration was measured by HPLC. 2.3.Microencapsulation process Microencapsulation of nisin was carried out by encapsulator Inotech IE-20 (Inotech Biosystems International, Inc) using 300 µm nozzle and syringe pump. Different ratios of sodium alginate (0.5, 1 and 2 %) were mixed with resistant gelatinized or non-gelatinized resistant starch to form seven different combinations of polymer mixtures. In addition, the formulas composed of 1 % alginate or 0.5 % were used as a control (Table 1). Nisin was added at a final concentration of 100 µg / ml
polymer. The resulting solution was added dropwise into a 5 % of sterilized calcium chloride solution at pH 6.00 using the encapsulator with a flow rate 2.9 ml/min, vibration frequency 2000 Hz. The resulting beads were continually stirred at 300 RPM for 4h at room temperature, Then Nisin-loaded beads were kept at 4 ˚C until using. 2.4. Morphological characterization of microcapsules
Beads were examined directly after formation for their size, integrity and homogeny under light microscope at 40 x magnification power. Observations of the external bead structure were made by macrophotographs using a Microscope Olympus BX51 (Center Valley, PA, USA) (software cellSens) The internal structure of selected formulas was examined by transmission electron microscopy (TEM). The microcapsules were fixed using 2.5% glutaraldehyde and mounted on metal grids. The samples were stained for one minute with uranyl acetate and then rinsed by immersion in deionized water and finally, dried with filter paper. Microcapsules observations were performed at high resolution (80 KV) with a JEOL 1200EX electron microscope (JEOL Ltd., Akishima, Japan). 2.5. Fourier Transform-Infrared (FTIR) Spectroscopy.
The FTIR examination was done on the different formulas of alginate / starch microparticles. The microcapsules were dried at room temperature for 24 h. The KBR desks were prepared by mixing 1 mg of sample and 100 mg of ground KBr and pressing it to form a pellet at 10 tons. The pellets were placed in the sample holder and spectral scanning was carried out using FTIR spectrometer (FTIR Nicolet 6700, Thermos Scientific) with a deuterated triglycine sulfate (DTGS) detector. The system is continuously purged with dried air to prevent the interference of water vapor. Spectra were acquired in transmission mode. A total of 256 scans was accumulated for each sample in the mid-IR region (400 — 4000 cm-1) with a resolution of 4 cm−1. The spectra were baseline corrected
and
normalized.
Data
processing
was
done
using
the
Omnicâ
software,
Version 9.1, Thermo Scientific, Madison, WI, USA 2.6.Antimicrobial activity of encapsulated nisin Antimicrobial activity of encapsulated nisin was determined directly using the agar diffusion assay. Where, 0.1 g from each capsule was added directly to an agar diffusion plate containing Pediococcus acidilactici UL5 as indicator strain. After 18 h of incubation at 37 °C, the inhibition zone was measured. A standard curve was prepared using the same technique of using serial dilution from free nisin. 2.7.Encapsulation efficiency (EE) The encapsulation efficacy (EE) was defined as the percentage of the nisin trapped in the microcapsules to the initial amount of nisin added to the polymer mixtures. The nisin beads were dissolved with equal volume of 0.1 Mol / L phosphate buffer solution at pH 8.00 under 60 ºC and stirred at 200 RPM until the complete dissolution of the alginate beads. The complete dissolution of the beads was confirmed by a light microscope. The microbiological activity of the nisin released from the dissolved beads was determined by the agar diffusion assay as described before. Where, 80 µl of each dissolved bead were added to an agar diffusion plate. The initial dilution of disaggregation was calculated to determine the percentage of EE. 2.8.Determination of nisin release The microcapsules were distributed in Falcone tube containing 20 ml of skim milk media (12% w/v) and incubated at 4 ˚C with a frequent shaking each day. Samples were taken weekly for one month to determine the nisin activity using the agar diffusion assay as described previously. 2.9 Application of encapsulated Nisin in cheese slurry
The cheddar cheese powder was prepared as described by Gardner-Fortier, St-Gelais, Champagne and Vuillemard [28] using pasteurized whole milk (Laiterie Chalifoux Inc., Sorel-Tracy, QC, Canada). The milk was acidified by glucono-δ-lactone (Sigma, St-Louis, MO, USA). Then, the curd was lyophilized. The lyophilized starter-free cheese was reduced to powder using a disc grinder Quadro ComIL (Waterloo, ON, Canada), then irradiated at 5 kGy with Cobalt 60 for 2 h. The cheddar cheese powder was divided into two groups of 100 g each. The cheese slurry was prepared at a chemical composition mimic to cheddar cheese, with pH value of 5.3 adjusted using lactic acid solution, 2 % salt, 32 % fat and 38 % humidity. Both groups were inoculated with cheddar cheese starter at 8 log cfu/g culture and C. tyrobutyricum ATTC 25755 at 4 log cfu/g and one group containing encapsulated nisin at concentration 2.44 µg nisin/ g cheese. Both groups were incubated at 4 ˚C for four weeks. Samples were taken to determine the C. tyrobutyricum count on weekly basis.
3. Results and discussion 3.1.Microscopic examination of microcapsules: Microscopic characterization of alginate/ gelatinized or non-gelatinized starch microcapsules showed in Fig 1. The image showed that the micro capsules have an integer spherical shape without any cracks, homogenous size. The average size of capsules is 300 ± 20 µm for formulas containing gelatinized starch and 270 ± 20 µm for formulas containing non-gelatinized starch. Moreover, the particles of non-gelatinized starch were visible inside the capsules, while starch was not visible in the capsules that containing gelatinized starch. This observation is in agreement with the previous studies [18, 29-31]. The spherical shape formation of microcapsules was in contrast to the drop
shape obtained by Maresca, De Prisco, La Storia, Cirillo, Esposito and Mauriello [16] who reported that, this shape is due to the electrostatic interaction between nisin and alginate matrices. Experiments were performed in triplicates beads sizes were reported as average ± standard error. 3.2.Fourier Transform-Infrared (FTIR) spectroscopy FTIR Spectrum of alginate beads (Figure 2) showed band in the range of 3600-3000 cm-1 corresponded to OH stretching vibration. Absorption bands in the range of 2900–2700 cm−1 are assigned to stretching vibrations of aliphatic C–H groups. Bands Observed at 1592 cm-1 and 1415 cm-1 are attributed to asymmetric and symmetric stretching modes of COO-, respectively. These bands are showing that the carboxylate groups of alginate are in the form of carboxylate ions (COO-) and were commonly used to distinguish between alginate and its other products (Daemi and Barikani, 2012). The complex bands in the region 1200̻ 800 cm−1 are attributed to̻ C̻ O and C̻ O̻ C stretching vibrations that are considered as the fingerprinting area characterize the natural polysaccharides. The strong bands at 1027 cm−1 were attributed to the C–O stretching vibration of pyranosyl ring and the C–O stretching, respectively, with contributions from the deformation modes of C–C–H and C–O–H (Atia et al. 2016). The presence of strong asymmetric stretching absorption band at 1614 cm−1, and weaker symmetric stretching band near 1418 cm−1 supported the presence carboxylate anion of alginate structure. The addition of resistant starch to the alginate did not cause a red-shift in the alginate wavenumbers of 1614 and 1418 cm-1 which clarifies that alginate did not interact and form hydrogen bonding with starch under the preparation of microcapsules with the ionotropic method. In literature, the preparation of alginate starch beads by emulsion methodology involved interactions of the two polymers with hydrogen bonding [26]. The nisin loaded starch̻ alginate beads had the significant characters of alginate and starch in the FTIR spectrum, however, the addition of nisin caused a shift and narrowing of the OH stretching
band at 3388 cm-1 suggesting hydrogen bonding interactions between the polymers and nisin. The FTIR analysis confirmed the compatibility of the nisin with sodium alginate and starch in starch̻ alginate beads prepared by ionotropic gelation technique. 3.3.Antimicrobial activity of encapsulated nisin The antibacterial activity of microencapsuled nisin was examined directly after preparation in the agar diffusion plate using Pediococcus acidilactici UL5 as an indicator strain. Figure 3 present the inhibition zone of nisin beads in the agar diffusion plate against the Pediococcus acidilactici UL5 after 18h of incubation at 37 ˚C. All microcapsules formulas showed activity against Pediococcus acidilactici UL5. No significant differences in the inhibition zone were obtained among the different formulas. The inhibition zone produced by all formulas showed relatively the same size of inhibition zone of 15 ± 2 mm.
3.4.Encapsulation efficiency (EE) Out of the ten formulas listed in Table 1, seven formulas were selected. The high viscosity of the polymer mixture that containing 2% of gelatinized starch or alginate in addition to the high concentration of starch particles in formula containing 1% alginate and 1% of non-gelatinized starch, prevented from obtaining the suitable capsule size from these formulas. In addition, these formulas were not suitable for large scale production of microcapsules. As such the formulas H, I and G in Table 1 were not further considered for the analysis. The encapsulation efficiency of the different formulas presented in Tabe 2. The
formulas composed from 1 % alginate / 0.5% non-gelatinized starch and 1% alginate/ 0.5 % gelatinized starch showed the highest encapsulation efficiency approximately 33.27 % and 33.12 %, respectively compared to 7.77 % and 24.3% EE in case of 0.5 and 1 % of control alginate beads, respectively. Which indicated that, the presence of starch in the encapsulation matrix increased the EE of the microcapsules. These results are in agreement with Sultana, Godward, Reynolds,
Arumugaswamy, Peiris and Kailasapathy [32]. The authors reported that, the presence of Himaize resistant starch with alginate for microencapsulation of probiotic bacteria increased the EE using the w /o emulsion method which produce particle size up to 500 µm [32]. Moreover, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] found that the addition of 2 % of resistant starch to alginate matrix increase the EE of nisin using the emulsion method due to the stabilization effect of resistant starch in alginate. On the other hand, the increase in the starch percentage, cause lower EE in both gelatinized and nongelatinized starch. This may be due to the space occupied by the starch particles inside the capsules that reduces the amount of encapsulated nisin under the fixed particle size obtained using the employed vibrating methodology. Our values showed lower EE values compared to other studies that used emulsification encapsulation technology for nisin encapsulation [15, 16, 26, 33].
3.5.Determination of nisin release Before the application of the nisin-loaded microcapsules in food products, the release of nisin from microcapsules was evaluated in a skim milk media at 4 ˚C for one month in order to select the best formulas for large scale production. The release of nisin from microparticles was measured according to Maresca, De Prisco, La Storia, Cirillo, Esposito and Mauriello [16] with some modifications. The antimicrobial activity of the released nisin from microcapsules in the skim milk media was measured by the agar diffusion assay using Pediococcus acidilactici UL5 as indicator strain. Table 3 presents the amount of released nisin from the microcapsules in skim milk media for one month. The results show that, all the formulas exhibit stable and gradual release during first ten days in skim milk media. The same behavior was observed using alginate beads by vibrating technology. Where, the antimicrobial activity of microencapsulated nisin was stable
during 168 h of incubation in 1:1 Ringer solution at pH 6.00 at 4 ˚C using Brochothrix thermosphacta 7R1 as indicator strain (Maresca, De Prisco et al. 2016). Also, Chakraborty, Khandai, Sharma, Khanam, Patra, Dinda and Sen [18] found that, the addition of pectin to form sodium- alginate pectin formula, to encapsulated the aceclofenac drug, helped to increase the in vitro gradual release using phosphate buffer at pH 6.8 at 37 ˚C to mimic the intestinal condition during 12 hours compared to formulas containing alginate alone, which start in erosion before the complete swelling which led to a faster drug release during the first four hours of incubation [18]. Moreover, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, ShojaeeAliabadi and Khaksar [26] observe that the time needed to release the 50 % of the amount of loaded nisin from alginate -resistant starch microcapsules prepared by the w/o emulsion method is 168 hours compared to 96 hours in the case of alginate microcapsules in distilled water at room temperature under static condition. Which indicated that, the release of nisin from starch- alginate microcapsules is gradual and under controlled manner [26]. On the other hand, after eleven days of incubation there is a reduction in the nisin amount that released from all formulas and the highest reduction was observed in the control formulas (0.5 and 1 %alginate). The best formulas were 10.5 % alginate-non-gelatinized starch and 1-1 % alginate -gelatinized starch. Where, these capsules were able to protect and gradually release the nisin for one month. The experiment extended for another month under the same condition. Only the skim milk supernatant from the formula 1 % alginate / 0.5 % non-gelatinized starch had activity in the agar diffusion plate. For that, this formula was selected due to its high encapsulation efficiency and the gradual release and protection of nisin during two months of storage. This study is the first study that determined the microcapsules stability during long term of incubation of two months in skim milk media. 3.6.Transmission electron microscope (TEM):
The internal structure of the microcapsule composed of 1: 0.5 sodium alginate: none-gelatinized starch was examined directly after preparation.
Figure 4 shows the middle core of the
microcapsules and their exterior wall. The photomicrograph illustrates that, the wall of microcapsules composed of an outer layer from sodium alginate and internal or parallel layer from non-gelatinized starch. Which is in agreement with other studies, that confirmed the presence of other polymer with alginate reduce the pore size of alginate which regulates the release of bioactive molecules [18, 26]. Also, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] reported that, the presence of resistant starch in the alginate microcapsules formed a compact, spherical with no cracks microcapsules compare to microcapsules composed of alginate alone using scanning electron microscope. The core of the beads is presented in Figure 4B. The core is homogeneous with no wall layers presented that facilitate the nisin entrapment within the beads. 3.7.Application of encapsulated nisin in Cheese: The addition of encapsulated nisin (E-Nisin) to cheese slurry did not have a significant effect on the cheddar cheese starter count (Figure 5.A). On the other hand, a significant reduction in C. tyrobutyricum count obtained in case of the E-Nisin compared with the control. That reduction was approximately 1.4 log after one week of incubation at 4 ˚C. Moreover, starting from the second week until week four, C. tyrobutyricum was completely inhibited and not detected in the cheese samples. These results indicated that, the encapsulated nisin has an inhibition effect of the growth of C. tyrobutyricum in cheddar cheese at the large-scale production. The same inhibitory effect was observed in a previous study against C. tyrobutyricum CECT 4011 using L. lactis IPLA 729 strain that produces nisin Z in a combination with starter culture during the manufacturing of Vidiago cheese, a semi-hard farmhouse variety, produced in Asturias, Spain. Where, the counts of
C. tyrobutyricum were reduced by 3 logs at nisin concentration of 1600 AU / ml compared to an increase in the count of the control group of 3.2 logs during the 30 days of storage at 12 ˚C [34]. Moreover, encapsulated nisin z in liposomes at concentration 300 IU / g reduce the count of Listeria innocua ATCC 33090 by 3 logs immediately after a cheddar cheese production, while, after 6 months of the cheddar cheese storage, the L. innocua counts were less than 10 cfu/ g cheese and 90% of the encapsulated nisin was still active [35]. 4. Conclusion Nisin was encapsulated by five different ratios of alginate/ gelatinized or non-gelatinized resistant starch. Microcapsules appeared under light microscope in spherical shape, homogenized size and integrity. The presence of resistant starch at a concentration of 0.5 % increased the EE compared to formula of alginate alone. Also, the blending of resistant starch with alginate extended the gradual release and activity of nisin during storage. Finally, the results proved that the alginate/ non-gelatinized resistant starch microcapsule is a suitable carrier for the protection and controlled release of nisin in food applications.
5. References: [1] M.E. Stiles, Biopreservation by lactic acid bacteria, Antonie van leeuwenhoek 70(2-4) (1996) 331-345. [2] P.M. Davidson, F.J. Critzer, T.M. Taylor, Naturally occurring antimicrobials for minimally processed foods, Annual Review of Food Science and Technology 4 (2013) 163-190. [3] A. Galvez, M. Burgos, R. Lopez, R. Pulido, Natural antimicrobials for food biopreservation. Food biopreservation (pp 3–11), Springer: New York, Heidelberg, Dordrecht, London, 2014. [4] P. García, L. Rodríguez, A. Rodríguez, B. Martínez, Food biopreservation: promising strategies using bacteriocins, bacteriophages and endolysins, Trends in food science & technology 21(8) (2010) 373-382. [5] C.-I. Cheigh, Y.-R. Pyun, Nisin biosynthesis and its properties, Biotechnology letters 27(21) (2005) 1641-1648. [6] Y. Chi-Zhang, K.L. Yam, M.L. Chikindas, Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system, International journal of food microbiology 90(1) (2004) 15-22. [7] L.J. de Arauz, A.F. Jozala, P.G. Mazzola, T.C.V. Penna, Nisin biotechnological production and application: a review, Trends in food science & technology 20(3-4) (2009) 146-154.
[8] M. Lotfi, H. Tajik, M. Moradi, M. Forough, E. Divsalar, B. Kuswandi, Nanostructured chitosan/monolaurin film: Preparation, characterization and antimicrobial activity against Listeria monocytogenes on ultrafiltered white cheese, Lwt 92 (2018) 576-583. [9] K.M. Soto, M. Hernández-Iturriaga, G. Loarca-Piña, G. Luna-Bárcenas, S. Mendoza, Antimicrobial effect of nisin electrospun amaranth: pullulan nanofibers in apple juice and fresh cheese, International journal of food microbiology 295 (2019) 25-32. [10] D. Xiao, P.M. Davidson, Q. Zhong, Release and antilisterial properties of nisin from zein capsules spray-dried at different temperatures, LWT-Food Science and Technology 44(10) (2011) 1977-1985. [11] H. Cui, Y. Dai, L. Lin, Enhancing antibacterial efficacy of nisin in pork by poly-γ-glutamic acid/poly-llysine nanoparticles encapsulation, Journal of food safety 38(4) (2018) e12475. [12] H. Cui, J. Wu, C. Li, L. Lin, Anti-listeria effects of chitosan-coated nisin-silica liposome on Cheddar cheese, Journal of dairy science 99(11) (2016) 8598-8606. [13] H. Cui, J. Wu, C. Li, L. Lin, Improving anti-listeria activity of cheese packaging via nanofiber containing nisin-loaded nanoparticles, LWT-Food Science and Technology 81 (2017) 233-242. [14] R. Laridi, E. Kheadr, R.-O. Benech, J. Vuillemard, C. Lacroix, I. Fliss, Liposome encapsulated nisin Z: optimization, stability and release during milk fermentation, International dairy journal 13(4) (2003) 325-336. [15] R. Khaksar, S.M. Hosseini, H. Hosseini, S. Shojaee-Aliabadi, M.A. Mohammadifar, A.M. Mortazavian, K. khosravi-Darani, N. Haji Seyed Javadi, R. Komeily, Nisin-loaded alginate-high methoxy pectin microparticles: preparation and physicochemical characterisation, International journal of food science & technology 49(9) (2014) 2076-2082. [16] D. Maresca, A. De Prisco, A. La Storia, T. Cirillo, F. Esposito, G. Mauriello, Microencapsulation of nisin in alginate beads by vibrating technology: Preliminary investigation, LWT-Food Science and Technology 66 (2016) 436-443. [17] E.-S. Chan, S.-L. Wong, P.-P. Lee, J.-S. Lee, T.B. Ti, Z. Zhang, D. Poncelet, P. Ravindra, S.-H. Phan, Z.-H. Yim, Effects of starch filler on the physical properties of lyophilized calcium–alginate beads and the viability of encapsulated cells, Carbohydrate Polymers 83(1) (2011) 225-232. [18] S. Chakraborty, M. Khandai, A. Sharma, N. Khanam, C. Patra, S. Dinda, K. Sen, Preparation, in vitro and in vivo evaluation of algino-pectinate bioadhesive microspheres: An investigation of the effects of polymers using multiple comparison analysis, Acta pharmaceutica 60(3) (2010) 255-266. [19] N. Lin, J. Huang, P.R. Chang, L. Feng, J. Yu, Effect of polysaccharide nanocrystals on structure, properties, and drug release kinetics of alginate-based microspheres, Colloids and Surfaces B: Biointerfaces 85(2) (2011) 270-279. [20] Y. Dumoulin, L.H. Cartilier, M.A. Mateescu, Cross-linked amylose tablets containing α-amylase: an enzymatically-controlled drug release system, Journal of Controlled Release 60(2-3) (1999) 161-167. [21] M. Santander-Ortega, T. Stauner, B. Loretz, J.L. Ortega-Vinuesa, D. Bastos-González, G. Wenz, U.F. Schaefer, C.-M. Lehr, Nanoparticles made from novel starch derivatives for transdermal drug delivery, Journal of Controlled Release 141(1) (2010) 85-92. [22] A.E.-H. Ali, A. AlArifi, Characterization and in vitro evaluation of starch based hydrogels as carriers for colon specific drug delivery systems, Carbohydrate Polymers 78(4) (2009) 725-730. [23] J. Alias, I. Silva, I. Goni, M. Gurruchaga, Hydrophilic amylose-based graft copolymers for controlled protein release, Carbohydrate Polymers 74(1) (2008) 31-40. [24] A.C. Freire, C.C. Fertig, F. Podczeck, F. Veiga, J. Sousa, Starch-based coatings for colon-specific drug delivery. Part I: The influence of heat treatment on the physico-chemical properties of high amylose maize starches, European Journal of Pharmaceutics and Biopharmaceutics 72(3) (2009) 574-586. [25] I. Silva, M. Gurruchaga, I. Goñi, Physical blends of starch graft copolymers as matrices for colon targeting drug delivery systems, Carbohydrate Polymers 76(4) (2009) 593-601.
[26] S.M. Hosseini, H. Hosseini, M.A. Mohammadifar, J.B. German, A.M. Mortazavian, A. Mohammadi, S. Shojaee-Aliabadi, R. Khaksar, Preparation and characterization of alginate and alginate-resistant starch microparticles containing nisin, Carbohydrate Polymers 103 (2014) 573-580. [27] A. Okereke, T.J. Montville, Bacteriocin inhibition of Clostridium botulinum spores by lactic acid bacteria, Journal of food protection 54(5) (1991) 349-353. [28] C. Gardner-Fortier, D. St-Gelais, C.P. Champagne, J.-C. Vuillemard, Determination of optimal conditions for γ-aminobutyric acid production by Lactococcus lactis ssp. lactis, International dairy journal 32(2) (2013) 136-143. [29] S. Jana, S.A. Ali, A.K. Nayak, K.K. Sen, S.K. Basu, Development of topical gel containing aceclofenaccrospovidone solid dispersion by “Quality by Design (QbD)” approach, Chemical Engineering Research and Design 92(11) (2014) 2095-2105. [30] G.M. Fujiwara, R. Campos, C.K. Costa, J.d.F.G. Dias, O.G. Miguel, M.D. Miguel, F.d.A. Marques, S.M.W. Zanin, Production and characterization of alginate-starch-chitosan microparticles containing stigmasterol through the external ionic gelation technique, Brazilian Journal of Pharmaceutical Sciences 49(3) (2013) 537-547. [31] A.K. Nayak, J. Malakar, D. Pal, M.S. Hasnain, S. Beg, Soluble starch-blended Ca2+-Zn2+-alginate composites-based microparticles of aceclofenac: Formulation development and in vitro characterization, Future Journal of Pharmaceutical Sciences 4(1) (2018) 63-70. [32] K. Sultana, G. Godward, N. Reynolds, R. Arumugaswamy, P. Peiris, K. Kailasapathy, Encapsulation of probiotic bacteria with alginate–starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt, International journal of food microbiology 62(1-2) (2000) 47-55. [33] K. Narsaiah, S.N. Jha, R.A. Wilson, H.M. Mandge, M.R. Manikantan, Optimizing microencapsulation of nisin with sodium alginate and guar gum, Journal of food science and technology 51(12) (2014) 40544059. [34] N. Rilla, B. Martı ́nez, T. Delgado, A. Rodrı ́guez, Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer, International journal of food microbiology 85(1-2) (2003) 23-33. [35] R.-O. Benech, E. Kheadr, R. Laridi, C. Lacroix, I. Fliss, Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture, Appl. Environ. Microbiol. 68(8) (2002) 3683-3690.
Legend of Tables: Table 1 : the composition of different microcapsules. Table 2 : encapsulation efficiency of different microcapsules. Table 3: the release of nisin (µg/ ml) from microcapsules during 26 days in skim milk media
Legend of Figures:
Figure 1 shows the macrophotographs of nisin loaded beads prepared with different concentration of alginate/ gelatinized or non-gelatinized starch: A: alginate 0.5%; A2) 0.51%alginate/ G-starch; A3) 0.5- 1%alginate/ non-starch and B1) alginate 1%; B2) 1- 0.5% alginate/ non-starch; B3) 1- 0.5% alginate/ G-starch; B4) 1- 1% alginate/ G-starch. Describe precisely the pictures. Figure 2: FTIR spectra of Alginate, starch, Alginate-starch microparticles and nisin-loaded Alginate-starch microparticles. Figure 3: Antimicrobial activity of microencapsulated nisin directly using the agar diffusion assay and Pediococcus acidilactici UL5 as indicator strain. Figure 4: Transmission electron micrograph of 1- 0.5% alginate/ non-starch microcapsule. A) microcapsule wall and b) the interior of a microcapsule. Figure 5: Effect of encapsulated nisin on the starter culture and C. tyrobutyricum over the period of 4 weeks.
Table 1: The composition of different microcapsules. Formulas A B C D E F G H I J
Sodium- Alginate % 0.5 1
Resistant starch % 0 0
0.5
1
1
0.5
1
1
2
2
Table 2: Encapsulation efficiency of different microcapsules.
Formulas
EE %
Alginate 0.5% Alg – St 0.5 – 1 G Alg – St 0.5 – 1 Non Alginate 1% Alg – St 1 – 0.5 Non Alg – St 1 – 0.5 G Alg – St 1–1G
7.77±1.2 12.21±1.4 10.98±0.7 24.3±1.1 33.27±1.4 33.12±1.2 20.61±0.8
Starch Physical stat -------Gelatinized Non- gelatinized Gelatinized Non- gelatinized Gelatinized Non- gelatinized Gelatinized Non- gelatinized
Table 3: the release of nisin (µg/ml) from microcapsules during 26 days in skim milk media Days
AL 0.5%
1 2 3 4 5 6 7 10 13 16 19 23 26 60
3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 2.84±0.1 1.45±0.1 0.07±0.1 ND
AL-ST 0.5-1 NON 3.97 2.84 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 1.45 ND
AL-ST 10.5 G
AL-ST 0.5-1 G
AL-ST 11G
AL-ST 10.5 NON
AL 1%
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.03 1.04 ND
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 ND
3.97 3.97 3.97 3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 ND
3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 2.84 2.00
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.03 0.10 0.10 ND
A1
A3
A2
B1
B2
B3
B4
Figure 1 shows the macrophotographs of nisin loaded beads prepared with different concentration of alginate/ gelatinized or non-gelatinized starch: A: alginate 0.5%; A2) 0.5- 1% alginate/ G-starch; A3) 0.5- 1% alginate/ non-starch and B1) alginate 1%; B2) 1- 0.5% alginate/ non-starch; B3) 10.5% alginate/ G-starch; B4) 1- 1% alginate/ G-starch.
0.60
Alginate 1%
NG ST 0.5%
Alg-NG ST 1-0.5%
Alg-NG ST 1-0.5 Nisin
1592
0.55
0.50
1027
0.45 1415
Absorbance
0.40 0.35 0.30
0.25 0.20 0.15 0.10 0.05
0.00 4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Figure 2: FTIR spectra of Alg, starch, Alg-starch microparticles and nisin-loaded Alg-starch microparticles. *NG ST = non-gelatinized starch
AL 0.5% 15mm
AL 1% 15 mm
AL-S 0.5-1 G% 17 mm
AL- S 1- 0.5 G 15 mm
AL-S 0.5-1 NON% 17 mm
AL- S 1- 1 G 16 mm
Figure 3: Antimicrobial activity of microencapsulated nisin directly using the agar diffusion assay and Pediococcus acidilactici UL5 as indicator strain.
A
B
Figure 4: Transmission electron micrograph of 1- 0.5%alginate/ non gelatinized-starch microcapsule. A) microcapsule wall and b) the middle of microcapsule.
cfu/g
Starter Culture 8.8 8.6 8.4 8.2 8 7.8 7.6 7.4 7.2 7
Control (+) E-Nisin T0
Week1
Week2
Week3
Week4
C. tyrobutyricum 4
cfu/g
3 Control (+) E-Nisin
2 1
0 T0
Week1
Week2
Week3
Week4
Figure 5: Effect of encapsulated nisin on the starter culture and C. tyrobutyricum over the period of 4 weeks.
S0141-8130(19)35976-8 https://doi.org/10.1016/j.ijbiomac.2019.10.248 BIOMAC 13749
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
30 July 2019 24 October 2019 26 October 2019
Please cite this article as: H. Hassan, A. Gomaa, M. Subirade, E. Kheadr, D. St-Gelais, I. Fliss, Novel design for alginate/resistant starch microcapsules controlling nisin release, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.248
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© 2019 Published by Elsevier B.V.
Novel design for alginate/ resistant starch microcapsules controlling nisin release Hebatoallah Hassan a,b,c, Ahmed Gomaa a,d1, Muriel Subirade a, Ehab Kheadr c, Daniel St-Gelais e & Ismail Fliss a1 a STELA
Dairy Research Center, Food sciences department, Institute of Nutrition and Functional Foods (INAF), Université Laval, Québec, Canada b Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt c Faculty of Agriculture, Functional Foods and Nutraceuticals Lab, Alexandria University, Alexandria, Egypt d Nutrition and Food Science Department, National Research Center, Cairo, Egypt e Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Canada
Abstract This study aimed to develop a novel nontoxic, biocompatible, biodegradable, and cost-efficient matrix for the encapsulation of antimicrobial component (nisin) to be used as bio-preservative agent in cheddar cheese. Nisin A loaded beads were prepared from alginate at 0.5 %, 1% and 2 %; and hi-maize resistant starch at 0.5 or 1 %. Beads were characterized by microscopic examination and transmission electron microscopy. Molecular structures were investigated by FTIR, and particle size distributions were measured. The Entrapment efficiency (EE) was measured microbiologically by agar diffusion. The encapsulated nisin showed similar inhibition activities in all developed formulas with an inhibition zone of 15±2 mm. The FTIR analysis confirmed the compatibility of the nisin with sodium alginate and starch. The formulas composed of 1 % Alginate and 0.5 % Non-Gelatinized Starch had the highest encapsulation efficiency among other formulas (33 %). Moreover, that formula allowed the protection and gradual release of the encapsulated
1
Corresponding author: Institute INAF, Laval University, Pavillon Paul-Comtois, Québec, Canada. Fax : +1 (418) 656-3353; E-mail : [email protected]
nisin during a long-term storage for up to two months. Application in cheddar cheese proved the inhibition of encapsulated nisin on the growth of C. tyrobutyricum at the large-scale production. In conclusion, the alginate (Alg)/ non-gelatinized resistant starch formula is suitable for the protection and controlled release of nisin in food applications. Keywords: Encapsulation; nisin; Cheddar cheese 1. Introduction Recently, there is an increasing interest in applying bio-preservatives in food products instead of chemical preservatives. Bio-preservation or biocontrol refers to the use of natural or controlled microbiota, or its antimicrobial products against food spoilage or pathogenic microbes to extend the shelf life and enhance the safety of foods [1]. According to Davidson et al., 2013 [2], the antimicrobial component should be nontoxic, effective at low concentrations in its natural form, economical to be applicable in commercial scale, also, does not affect the sensory parameters of the food product, in addition it inhibit a wide range of pathogenic and spoilage microorganisms [2]. Microbes usually live in complex ecosystems where they must interact with the surrounding components of the environment. They compete with each other for space and nutrients in order to survive. One of the most important competition strategies is based on the modification of the environment conditions by the release of antimicrobial substances as by-products of their normal metabolic activity that inhibit growth or even kill the competitors. It can be more specific encoded by specific genetic determinants aimed to combat against specific microbes [3]. lactic acid bacteria (LAB) have a great potential for extended use in bio-preservation. LAB exist naturally in many food systems and conceded as the cornerstone for developing fermented food
sector, also have a long history of safe use in fermented foods, and are classified as Generally Regarded as Safe (GRAS). During fermentation, LAB produce various antimicrobial compounds such as organic acids, diacetyl, hydrogen peroxide, bacteriocins or antifungal molecules [3]. Bacteriocins that are produced by lactic acid bacteria have a promising potential application as bio-preservatives. Although a great number of bacteriocins are discovered, nisin is the only one permitted for using in food industry [4]. Nisin is recognized as safe (GRAS) by FAO/WHO and Expert Committee on Food Additives. It has been used as bio-preservative in meat, vegetables and dairy products [5]. Nisin has a wide spectrum of antibacterial activity against gram positive bacteria and heat stable. However, nisin easily interacts with food ingredients like proteins, lipids and carbohydrates, which lead to a rapid inactivation. In addition, it is affected by the presence of proteolytic enzymes, which reduce its activity against food pathogenic or spoilage microorganisms [6, 7]. To overcome these limitations, encapsulation is a process that could be implemented in order to protect nisin from food matrix, provide and maintain the gradual release of nisin during food storage. Several matrices have been used in literatures for the encapsulation of nisin like liposomes (Laridi, Kheadr et al. 2003), alginate, chitosan or a mixture of polymers such as chitosan/ monolaurin [8]; amaranth / pullulan [9];zein capsules [10]; nanoparticles using poly-g-glutamic acid (g-PGA) and poly-Llysine (PLL) [11]; chitosan-coated nisin-silica [12] or polyethylene oxide nanofibers containing nisin-loaded poly-g-glutamic acid/chitosan. [13]. However, some drawbacks were attributed to these materials, including low encapsulation efficiency, instability, high cost and/or not applicable for large scale production. [8, 9, 14, 15]. In this study, we have used alginate and resistant starch to design a novel encapsulation matrix for nisin. Alginate is a biodegradable, food grade polymer, simple structure and easy to form capsules
in the presence of cations in aqueous solution [15, 16]. However, micro-particles produced from alginate alone exhibited physical instability, porous and hydrophilic structure which lead to rapid release of bioactive material [17, 18]. The additions of other materials like polysaccharides (starch, guar gum or pectin) , polysaccharide nano-crystals have been used to improve the stability and release of alginate [15, 19]. In another study, Chakraborty, Khandai, Sharma, Khanam, Patra, Dinda and Sen [18] used microcapsules from sodium alginate and pectin for encapsulation of anti-inflammatory drug to get sustained drug release in phosphate buffer at pH 6.8 for 12 hours compared to the microcapsules composed of sodium alginate alone. Due to the rigid coat that provided by pectin when it formed a gel in the presence of calcium during ion-exchange gelation. Starch is like alginate as a biodegradable polymer, nontoxic, biocompatible to food matrices and available at low cost [20]. Compared to native starch, resistant starch is resistant to enzymatic lysis which presents naturally in food matrices or produced by bacteria in case of fermented food products. Accordingly, resistant starch is stable during food storage and provide controlled release of bioactive material [20, 21]. Several studies used the starch and its derivatives like cross-linked starch,
starch-methacrylic
acid,
starch-ethylene
vinyl
alcohol
and
starch-2-
hydroxyethylmethacrylate as probiotics and peptide delivery system [22-25]. Recently, some studies have tried to encapsulate nisin for food applications using alginate alone with vibrating technology. Although the particle size achieved using vibrating technology are homogenic, the antimicrobial activity of nisin decreased after 168h at 4 ˚C and 72 hours at 20 ˚C [16]. Alginate and resistant starch polymers could form a promising mixture with the ability of largescale production for food application. Moreover, these mixtures are food grade microcapsules at
low cost. In literature there are very few studies that investigated alginate and starch. In this regard, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] used 2% of resistant starch with sodium alginate to encapsulate nisin by emulsion method. They found that, the presence of resistant starch increased the time of release of 50 % of the nisin up to one week longer than the formula composed of alginate alone. However, there is a gap in literature that needs more studies using different concentrations of starch and sodium alginate, in addition to, investigating the release during long period of storage in conditions mimic to food matrices. The aim of this study was to develop novel matrices for the encapsulation of antimicrobial component (nisin) to be used as bio-preservative agent for cheddar cheese. The encapsulation matrix was developed from alginate and resistant starch in order to improve the properties of each polymer and overcome the limitations. According to our knowledge, there are no previous studies about the production of microcapsules using alginate/ resistant starch by ionotropic gelation vibration technology. Also, the effect of the physical state of starch, gelatinized or non-gelatinized, on the structure and function properties of capsules has not been addressed. Moreover, no previous research investigated the effect of different combinations from alginate and starch at different concentrations in the characteristics of microcapsules. Finally, this study is the first study to investigate structure, stability, and storage of microcapsules during long term of storage. 2. Material and methods Resistant starch was obtained from Manitoba Strach products Inc, Canada. All bacterial culture media were purchased from Nutri Bact, Canada. Sodium alginate and calcium chloride were obtained from Sigma Aldrich, Canada. Cheddar cheese starter culture, L. lactis Subsp. cremoris
CUC222 and L. lactis Subsp. lactis CUC-H, were obtained from Agriculture and Agri-Food Canada, St-Hyacinthe, Canada. 2.1. Bacterial preparation Pediococcus acidilactici UL5 Strain obtained from Laval university culture collection. It was used as indicator strains in the nisin activity assay. Bacterial strain activated twice for 16 h at 37 ºC of incubation in MRS media before using. Spores production Clostridium tyrobutyricum ATTC 25755 was used. The strain was activated twice in Reinforced Clostridial Medium (RCM). Spores were prepared according to [27] with some modifications. The strain was inoculated at 1 % in RCM media and incubated two weeks at 37 ºC in anaerobic chamber. The spores were collected by centrifugation at 5000 g at 4 ºC for 10 min. the spores were resuspended in skim milk media (12%). The spores were enumerated using RCM media after heating at 85 ºC for 5 min to activate the germination of spores. 2.2.Nisin preparation Commercial nisin was obtained from Cayman chemical, USA. Commercial nisin was purified by Sep-Pack C18 column at 4 °C, at a flow rate of 3 ml/min. Followed by a concentration step by Speed-Vac for 6h at 50 °C. The nisin concentration was measured by HPLC. 2.3.Microencapsulation process Microencapsulation of nisin was carried out by encapsulator Inotech IE-20 (Inotech Biosystems International, Inc) using 300 µm nozzle and syringe pump. Different ratios of sodium alginate (0.5, 1 and 2 %) were mixed with resistant gelatinized or non-gelatinized resistant starch to form seven different combinations of polymer mixtures. In addition, the formulas composed of 1 % alginate or 0.5 % were used as a control (Table 1). Nisin was added at a final concentration of 100 µg / ml
polymer. The resulting solution was added dropwise into a 5 % of sterilized calcium chloride solution at pH 6.00 using the encapsulator with a flow rate 2.9 ml/min, vibration frequency 2000 Hz. The resulting beads were continually stirred at 300 RPM for 4h at room temperature, Then Nisin-loaded beads were kept at 4 ˚C until using. 2.4. Morphological characterization of microcapsules
Beads were examined directly after formation for their size, integrity and homogeny under light microscope at 40 x magnification power. Observations of the external bead structure were made by macrophotographs using a Microscope Olympus BX51 (Center Valley, PA, USA) (software cellSens) The internal structure of selected formulas was examined by transmission electron microscopy (TEM). The microcapsules were fixed using 2.5% glutaraldehyde and mounted on metal grids. The samples were stained for one minute with uranyl acetate and then rinsed by immersion in deionized water and finally, dried with filter paper. Microcapsules observations were performed at high resolution (80 KV) with a JEOL 1200EX electron microscope (JEOL Ltd., Akishima, Japan). 2.5. Fourier Transform-Infrared (FTIR) Spectroscopy.
The FTIR examination was done on the different formulas of alginate / starch microparticles. The microcapsules were dried at room temperature for 24 h. The KBR desks were prepared by mixing 1 mg of sample and 100 mg of ground KBr and pressing it to form a pellet at 10 tons. The pellets were placed in the sample holder and spectral scanning was carried out using FTIR spectrometer (FTIR Nicolet 6700, Thermos Scientific) with a deuterated triglycine sulfate (DTGS) detector. The system is continuously purged with dried air to prevent the interference of water vapor. Spectra were acquired in transmission mode. A total of 256 scans was accumulated for each sample in the mid-IR region (400 — 4000 cm-1) with a resolution of 4 cm−1. The spectra were baseline corrected
and
normalized.
Data
processing
was
done
using
the
Omnicâ
software,
Version 9.1, Thermo Scientific, Madison, WI, USA 2.6.Antimicrobial activity of encapsulated nisin Antimicrobial activity of encapsulated nisin was determined directly using the agar diffusion assay. Where, 0.1 g from each capsule was added directly to an agar diffusion plate containing Pediococcus acidilactici UL5 as indicator strain. After 18 h of incubation at 37 °C, the inhibition zone was measured. A standard curve was prepared using the same technique of using serial dilution from free nisin. 2.7.Encapsulation efficiency (EE) The encapsulation efficacy (EE) was defined as the percentage of the nisin trapped in the microcapsules to the initial amount of nisin added to the polymer mixtures. The nisin beads were dissolved with equal volume of 0.1 Mol / L phosphate buffer solution at pH 8.00 under 60 ºC and stirred at 200 RPM until the complete dissolution of the alginate beads. The complete dissolution of the beads was confirmed by a light microscope. The microbiological activity of the nisin released from the dissolved beads was determined by the agar diffusion assay as described before. Where, 80 µl of each dissolved bead were added to an agar diffusion plate. The initial dilution of disaggregation was calculated to determine the percentage of EE. 2.8.Determination of nisin release The microcapsules were distributed in Falcone tube containing 20 ml of skim milk media (12% w/v) and incubated at 4 ˚C with a frequent shaking each day. Samples were taken weekly for one month to determine the nisin activity using the agar diffusion assay as described previously. 2.9 Application of encapsulated Nisin in cheese slurry
The cheddar cheese powder was prepared as described by Gardner-Fortier, St-Gelais, Champagne and Vuillemard [28] using pasteurized whole milk (Laiterie Chalifoux Inc., Sorel-Tracy, QC, Canada). The milk was acidified by glucono-δ-lactone (Sigma, St-Louis, MO, USA). Then, the curd was lyophilized. The lyophilized starter-free cheese was reduced to powder using a disc grinder Quadro ComIL (Waterloo, ON, Canada), then irradiated at 5 kGy with Cobalt 60 for 2 h. The cheddar cheese powder was divided into two groups of 100 g each. The cheese slurry was prepared at a chemical composition mimic to cheddar cheese, with pH value of 5.3 adjusted using lactic acid solution, 2 % salt, 32 % fat and 38 % humidity. Both groups were inoculated with cheddar cheese starter at 8 log cfu/g culture and C. tyrobutyricum ATTC 25755 at 4 log cfu/g and one group containing encapsulated nisin at concentration 2.44 µg nisin/ g cheese. Both groups were incubated at 4 ˚C for four weeks. Samples were taken to determine the C. tyrobutyricum count on weekly basis.
3. Results and discussion 3.1.Microscopic examination of microcapsules: Microscopic characterization of alginate/ gelatinized or non-gelatinized starch microcapsules showed in Fig 1. The image showed that the micro capsules have an integer spherical shape without any cracks, homogenous size. The average size of capsules is 300 ± 20 µm for formulas containing gelatinized starch and 270 ± 20 µm for formulas containing non-gelatinized starch. Moreover, the particles of non-gelatinized starch were visible inside the capsules, while starch was not visible in the capsules that containing gelatinized starch. This observation is in agreement with the previous studies [18, 29-31]. The spherical shape formation of microcapsules was in contrast to the drop
shape obtained by Maresca, De Prisco, La Storia, Cirillo, Esposito and Mauriello [16] who reported that, this shape is due to the electrostatic interaction between nisin and alginate matrices. Experiments were performed in triplicates beads sizes were reported as average ± standard error. 3.2.Fourier Transform-Infrared (FTIR) spectroscopy FTIR Spectrum of alginate beads (Figure 2) showed band in the range of 3600-3000 cm-1 corresponded to OH stretching vibration. Absorption bands in the range of 2900–2700 cm−1 are assigned to stretching vibrations of aliphatic C–H groups. Bands Observed at 1592 cm-1 and 1415 cm-1 are attributed to asymmetric and symmetric stretching modes of COO-, respectively. These bands are showing that the carboxylate groups of alginate are in the form of carboxylate ions (COO-) and were commonly used to distinguish between alginate and its other products (Daemi and Barikani, 2012). The complex bands in the region 1200̻ 800 cm−1 are attributed to̻ C̻ O and C̻ O̻ C stretching vibrations that are considered as the fingerprinting area characterize the natural polysaccharides. The strong bands at 1027 cm−1 were attributed to the C–O stretching vibration of pyranosyl ring and the C–O stretching, respectively, with contributions from the deformation modes of C–C–H and C–O–H (Atia et al. 2016). The presence of strong asymmetric stretching absorption band at 1614 cm−1, and weaker symmetric stretching band near 1418 cm−1 supported the presence carboxylate anion of alginate structure. The addition of resistant starch to the alginate did not cause a red-shift in the alginate wavenumbers of 1614 and 1418 cm-1 which clarifies that alginate did not interact and form hydrogen bonding with starch under the preparation of microcapsules with the ionotropic method. In literature, the preparation of alginate starch beads by emulsion methodology involved interactions of the two polymers with hydrogen bonding [26]. The nisin loaded starch̻ alginate beads had the significant characters of alginate and starch in the FTIR spectrum, however, the addition of nisin caused a shift and narrowing of the OH stretching
band at 3388 cm-1 suggesting hydrogen bonding interactions between the polymers and nisin. The FTIR analysis confirmed the compatibility of the nisin with sodium alginate and starch in starch̻ alginate beads prepared by ionotropic gelation technique. 3.3.Antimicrobial activity of encapsulated nisin The antibacterial activity of microencapsuled nisin was examined directly after preparation in the agar diffusion plate using Pediococcus acidilactici UL5 as an indicator strain. Figure 3 present the inhibition zone of nisin beads in the agar diffusion plate against the Pediococcus acidilactici UL5 after 18h of incubation at 37 ˚C. All microcapsules formulas showed activity against Pediococcus acidilactici UL5. No significant differences in the inhibition zone were obtained among the different formulas. The inhibition zone produced by all formulas showed relatively the same size of inhibition zone of 15 ± 2 mm.
3.4.Encapsulation efficiency (EE) Out of the ten formulas listed in Table 1, seven formulas were selected. The high viscosity of the polymer mixture that containing 2% of gelatinized starch or alginate in addition to the high concentration of starch particles in formula containing 1% alginate and 1% of non-gelatinized starch, prevented from obtaining the suitable capsule size from these formulas. In addition, these formulas were not suitable for large scale production of microcapsules. As such the formulas H, I and G in Table 1 were not further considered for the analysis. The encapsulation efficiency of the different formulas presented in Tabe 2. The
formulas composed from 1 % alginate / 0.5% non-gelatinized starch and 1% alginate/ 0.5 % gelatinized starch showed the highest encapsulation efficiency approximately 33.27 % and 33.12 %, respectively compared to 7.77 % and 24.3% EE in case of 0.5 and 1 % of control alginate beads, respectively. Which indicated that, the presence of starch in the encapsulation matrix increased the EE of the microcapsules. These results are in agreement with Sultana, Godward, Reynolds,
Arumugaswamy, Peiris and Kailasapathy [32]. The authors reported that, the presence of Himaize resistant starch with alginate for microencapsulation of probiotic bacteria increased the EE using the w /o emulsion method which produce particle size up to 500 µm [32]. Moreover, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] found that the addition of 2 % of resistant starch to alginate matrix increase the EE of nisin using the emulsion method due to the stabilization effect of resistant starch in alginate. On the other hand, the increase in the starch percentage, cause lower EE in both gelatinized and nongelatinized starch. This may be due to the space occupied by the starch particles inside the capsules that reduces the amount of encapsulated nisin under the fixed particle size obtained using the employed vibrating methodology. Our values showed lower EE values compared to other studies that used emulsification encapsulation technology for nisin encapsulation [15, 16, 26, 33].
3.5.Determination of nisin release Before the application of the nisin-loaded microcapsules in food products, the release of nisin from microcapsules was evaluated in a skim milk media at 4 ˚C for one month in order to select the best formulas for large scale production. The release of nisin from microparticles was measured according to Maresca, De Prisco, La Storia, Cirillo, Esposito and Mauriello [16] with some modifications. The antimicrobial activity of the released nisin from microcapsules in the skim milk media was measured by the agar diffusion assay using Pediococcus acidilactici UL5 as indicator strain. Table 3 presents the amount of released nisin from the microcapsules in skim milk media for one month. The results show that, all the formulas exhibit stable and gradual release during first ten days in skim milk media. The same behavior was observed using alginate beads by vibrating technology. Where, the antimicrobial activity of microencapsulated nisin was stable
during 168 h of incubation in 1:1 Ringer solution at pH 6.00 at 4 ˚C using Brochothrix thermosphacta 7R1 as indicator strain (Maresca, De Prisco et al. 2016). Also, Chakraborty, Khandai, Sharma, Khanam, Patra, Dinda and Sen [18] found that, the addition of pectin to form sodium- alginate pectin formula, to encapsulated the aceclofenac drug, helped to increase the in vitro gradual release using phosphate buffer at pH 6.8 at 37 ˚C to mimic the intestinal condition during 12 hours compared to formulas containing alginate alone, which start in erosion before the complete swelling which led to a faster drug release during the first four hours of incubation [18]. Moreover, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, ShojaeeAliabadi and Khaksar [26] observe that the time needed to release the 50 % of the amount of loaded nisin from alginate -resistant starch microcapsules prepared by the w/o emulsion method is 168 hours compared to 96 hours in the case of alginate microcapsules in distilled water at room temperature under static condition. Which indicated that, the release of nisin from starch- alginate microcapsules is gradual and under controlled manner [26]. On the other hand, after eleven days of incubation there is a reduction in the nisin amount that released from all formulas and the highest reduction was observed in the control formulas (0.5 and 1 %alginate). The best formulas were 10.5 % alginate-non-gelatinized starch and 1-1 % alginate -gelatinized starch. Where, these capsules were able to protect and gradually release the nisin for one month. The experiment extended for another month under the same condition. Only the skim milk supernatant from the formula 1 % alginate / 0.5 % non-gelatinized starch had activity in the agar diffusion plate. For that, this formula was selected due to its high encapsulation efficiency and the gradual release and protection of nisin during two months of storage. This study is the first study that determined the microcapsules stability during long term of incubation of two months in skim milk media. 3.6.Transmission electron microscope (TEM):
The internal structure of the microcapsule composed of 1: 0.5 sodium alginate: none-gelatinized starch was examined directly after preparation.
Figure 4 shows the middle core of the
microcapsules and their exterior wall. The photomicrograph illustrates that, the wall of microcapsules composed of an outer layer from sodium alginate and internal or parallel layer from non-gelatinized starch. Which is in agreement with other studies, that confirmed the presence of other polymer with alginate reduce the pore size of alginate which regulates the release of bioactive molecules [18, 26]. Also, Hosseini, Hosseini, Mohammadifar, German, Mortazavian, Mohammadi, Shojaee-Aliabadi and Khaksar [26] reported that, the presence of resistant starch in the alginate microcapsules formed a compact, spherical with no cracks microcapsules compare to microcapsules composed of alginate alone using scanning electron microscope. The core of the beads is presented in Figure 4B. The core is homogeneous with no wall layers presented that facilitate the nisin entrapment within the beads. 3.7.Application of encapsulated nisin in Cheese: The addition of encapsulated nisin (E-Nisin) to cheese slurry did not have a significant effect on the cheddar cheese starter count (Figure 5.A). On the other hand, a significant reduction in C. tyrobutyricum count obtained in case of the E-Nisin compared with the control. That reduction was approximately 1.4 log after one week of incubation at 4 ˚C. Moreover, starting from the second week until week four, C. tyrobutyricum was completely inhibited and not detected in the cheese samples. These results indicated that, the encapsulated nisin has an inhibition effect of the growth of C. tyrobutyricum in cheddar cheese at the large-scale production. The same inhibitory effect was observed in a previous study against C. tyrobutyricum CECT 4011 using L. lactis IPLA 729 strain that produces nisin Z in a combination with starter culture during the manufacturing of Vidiago cheese, a semi-hard farmhouse variety, produced in Asturias, Spain. Where, the counts of
C. tyrobutyricum were reduced by 3 logs at nisin concentration of 1600 AU / ml compared to an increase in the count of the control group of 3.2 logs during the 30 days of storage at 12 ˚C [34]. Moreover, encapsulated nisin z in liposomes at concentration 300 IU / g reduce the count of Listeria innocua ATCC 33090 by 3 logs immediately after a cheddar cheese production, while, after 6 months of the cheddar cheese storage, the L. innocua counts were less than 10 cfu/ g cheese and 90% of the encapsulated nisin was still active [35]. 4. Conclusion Nisin was encapsulated by five different ratios of alginate/ gelatinized or non-gelatinized resistant starch. Microcapsules appeared under light microscope in spherical shape, homogenized size and integrity. The presence of resistant starch at a concentration of 0.5 % increased the EE compared to formula of alginate alone. Also, the blending of resistant starch with alginate extended the gradual release and activity of nisin during storage. Finally, the results proved that the alginate/ non-gelatinized resistant starch microcapsule is a suitable carrier for the protection and controlled release of nisin in food applications.
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[26] S.M. Hosseini, H. Hosseini, M.A. Mohammadifar, J.B. German, A.M. Mortazavian, A. Mohammadi, S. Shojaee-Aliabadi, R. Khaksar, Preparation and characterization of alginate and alginate-resistant starch microparticles containing nisin, Carbohydrate Polymers 103 (2014) 573-580. [27] A. Okereke, T.J. Montville, Bacteriocin inhibition of Clostridium botulinum spores by lactic acid bacteria, Journal of food protection 54(5) (1991) 349-353. [28] C. Gardner-Fortier, D. St-Gelais, C.P. Champagne, J.-C. Vuillemard, Determination of optimal conditions for γ-aminobutyric acid production by Lactococcus lactis ssp. lactis, International dairy journal 32(2) (2013) 136-143. [29] S. Jana, S.A. Ali, A.K. Nayak, K.K. Sen, S.K. Basu, Development of topical gel containing aceclofenaccrospovidone solid dispersion by “Quality by Design (QbD)” approach, Chemical Engineering Research and Design 92(11) (2014) 2095-2105. [30] G.M. Fujiwara, R. Campos, C.K. Costa, J.d.F.G. Dias, O.G. Miguel, M.D. Miguel, F.d.A. Marques, S.M.W. Zanin, Production and characterization of alginate-starch-chitosan microparticles containing stigmasterol through the external ionic gelation technique, Brazilian Journal of Pharmaceutical Sciences 49(3) (2013) 537-547. [31] A.K. Nayak, J. Malakar, D. Pal, M.S. Hasnain, S. Beg, Soluble starch-blended Ca2+-Zn2+-alginate composites-based microparticles of aceclofenac: Formulation development and in vitro characterization, Future Journal of Pharmaceutical Sciences 4(1) (2018) 63-70. [32] K. Sultana, G. Godward, N. Reynolds, R. Arumugaswamy, P. Peiris, K. Kailasapathy, Encapsulation of probiotic bacteria with alginate–starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt, International journal of food microbiology 62(1-2) (2000) 47-55. [33] K. Narsaiah, S.N. Jha, R.A. Wilson, H.M. Mandge, M.R. Manikantan, Optimizing microencapsulation of nisin with sodium alginate and guar gum, Journal of food science and technology 51(12) (2014) 40544059. [34] N. Rilla, B. Martı ́nez, T. Delgado, A. Rodrı ́guez, Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer, International journal of food microbiology 85(1-2) (2003) 23-33. [35] R.-O. Benech, E. Kheadr, R. Laridi, C. Lacroix, I. Fliss, Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture, Appl. Environ. Microbiol. 68(8) (2002) 3683-3690.
Legend of Tables: Table 1 : the composition of different microcapsules. Table 2 : encapsulation efficiency of different microcapsules. Table 3: the release of nisin (µg/ ml) from microcapsules during 26 days in skim milk media
Legend of Figures:
Figure 1 shows the macrophotographs of nisin loaded beads prepared with different concentration of alginate/ gelatinized or non-gelatinized starch: A: alginate 0.5%; A2) 0.51%alginate/ G-starch; A3) 0.5- 1%alginate/ non-starch and B1) alginate 1%; B2) 1- 0.5% alginate/ non-starch; B3) 1- 0.5% alginate/ G-starch; B4) 1- 1% alginate/ G-starch. Describe precisely the pictures. Figure 2: FTIR spectra of Alginate, starch, Alginate-starch microparticles and nisin-loaded Alginate-starch microparticles. Figure 3: Antimicrobial activity of microencapsulated nisin directly using the agar diffusion assay and Pediococcus acidilactici UL5 as indicator strain. Figure 4: Transmission electron micrograph of 1- 0.5% alginate/ non-starch microcapsule. A) microcapsule wall and b) the interior of a microcapsule. Figure 5: Effect of encapsulated nisin on the starter culture and C. tyrobutyricum over the period of 4 weeks.
Table 1: The composition of different microcapsules. Formulas A B C D E F G H I J
Sodium- Alginate % 0.5 1
Resistant starch % 0 0
0.5
1
1
0.5
1
1
2
2
Table 2: Encapsulation efficiency of different microcapsules.
Formulas
EE %
Alginate 0.5% Alg – St 0.5 – 1 G Alg – St 0.5 – 1 Non Alginate 1% Alg – St 1 – 0.5 Non Alg – St 1 – 0.5 G Alg – St 1–1G
7.77±1.2 12.21±1.4 10.98±0.7 24.3±1.1 33.27±1.4 33.12±1.2 20.61±0.8
Starch Physical stat -------Gelatinized Non- gelatinized Gelatinized Non- gelatinized Gelatinized Non- gelatinized Gelatinized Non- gelatinized
Table 3: the release of nisin (µg/ml) from microcapsules during 26 days in skim milk media Days
AL 0.5%
1 2 3 4 5 6 7 10 13 16 19 23 26 60
3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 3.97±0.1 2.84±0.1 1.45±0.1 0.07±0.1 ND
AL-ST 0.5-1 NON 3.97 2.84 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 2.03 1.45 ND
AL-ST 10.5 G
AL-ST 0.5-1 G
AL-ST 11G
AL-ST 10.5 NON
AL 1%
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.03 1.04 ND
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 ND
3.97 3.97 3.97 3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 ND
3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 3.97 2.84 2.00
3.97 3.97 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.84 2.03 0.10 0.10 ND
A1
A3
A2
B1
B2
B3
B4
Figure 1 shows the macrophotographs of nisin loaded beads prepared with different concentration of alginate/ gelatinized or non-gelatinized starch: A: alginate 0.5%; A2) 0.5- 1% alginate/ G-starch; A3) 0.5- 1% alginate/ non-starch and B1) alginate 1%; B2) 1- 0.5% alginate/ non-starch; B3) 10.5% alginate/ G-starch; B4) 1- 1% alginate/ G-starch.
0.60
Alginate 1%
NG ST 0.5%
Alg-NG ST 1-0.5%
Alg-NG ST 1-0.5 Nisin
1592
0.55
0.50
1027
0.45 1415
Absorbance
0.40 0.35 0.30
0.25 0.20 0.15 0.10 0.05
0.00 4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Figure 2: FTIR spectra of Alg, starch, Alg-starch microparticles and nisin-loaded Alg-starch microparticles. *NG ST = non-gelatinized starch
AL 0.5% 15mm
AL 1% 15 mm
AL-S 0.5-1 G% 17 mm
AL- S 1- 0.5 G 15 mm
AL-S 0.5-1 NON% 17 mm
AL- S 1- 1 G 16 mm
Figure 3: Antimicrobial activity of microencapsulated nisin directly using the agar diffusion assay and Pediococcus acidilactici UL5 as indicator strain.
A
B
Figure 4: Transmission electron micrograph of 1- 0.5%alginate/ non gelatinized-starch microcapsule. A) microcapsule wall and b) the middle of microcapsule.
cfu/g
Starter Culture 8.8 8.6 8.4 8.2 8 7.8 7.6 7.4 7.2 7
Control (+) E-Nisin T0
Week1
Week2
Week3
Week4
C. tyrobutyricum 4
cfu/g
3 Control (+) E-Nisin
2 1
0 T0
Week1
Week2
Week3
Week4
Figure 5: Effect of encapsulated nisin on the starter culture and C. tyrobutyricum over the period of 4 weeks.