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Select a protective agent for encapsulation of Lactobacillus plantarum
Journal Pre-proof Select a protective agent for encapsulation of Lactobacillus plantarum Sumate Tantratian, Mathurose Pradeamchai PII:
S0023-6438(20)30063-3
DOI:
https://doi.org/10.1016/j.lwt.2020.109075
Reference:
YFSTL 109075
To appear in:
LWT - Food Science and Technology
Received Date: 15 October 2019 Revised Date:
9 December 2019
Accepted Date: 21 January 2020
Please cite this article as: Tantratian, S., Pradeamchai, M., Select a protective agent for encapsulation of Lactobacillus plantarum, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109075. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
CRediT author statement Sumate Tantratian: Writing-Reviewing and Editing, Supervision, Conceptualization, Data curation, Visualization Mathurose Pradeamchai: Investigation, Data curation, Formal Analysis
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Select a protective agent for encapsulation of Lactobacillus plantarum
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Sumate Tantratian1,3*and Mathurose Pradeamchai2
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1
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND
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2
Biotechnology Program, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND
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3
The development of foods and food additive from innovative microbial fermentation Research Group,
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Faculty of Science, Chulalongkorn University Bangkok 10330, THAILAND
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* corresponding author; [email protected]
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Abstract
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Five carbohydrates were applied as protective agents for encapsulation of Lactobacillus
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plantarum FT 35 which produced high acid and had ability to inhibit some pathogenic bacteria. They
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had glass transition temperatures in ascending order from glucose, sucrose, lactose, maltodextrin to
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soluble starch. The soluble starch demonstrated the best protection to cell during drying process and
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storage. The accretion of soluble starch higher than 2.5% in feed-in mixture for spray drying
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exhibited the increase in the viscosity of mixtures and the particle size of products. These products
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contained less number of viable cells and higher cell injury. The encapsulated products with 2.5%
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soluble starch exhibited a better storage stability than those with 1.5%. The encapsulated product was
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applied as a co-starter in pla-som fermentation without additional of carbon sources and also
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demonstrated fast acid production. The fermentation with encapsulated products displayed better
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inhibitory effect on inoculated pathogenic bacteria than indigenous fermentation. The soluble starch
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serves as a protective agent for encapsulated culture and also a good carbon source for lactic acid
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bacteria in the fermentation of pla-som.
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Keywords; glass transition temperature, carbohydrate, encapsulation, carbon source, pla-som
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fermentation
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Introduction
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Encapsulation is the process to protect bacterial cell or bioactive substance against harsh
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environment in wall materials (Arpagaus et al., 2018). It allow probiotics to be widely applied in
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fresh or dried food markets (Martín et al., 2015). Spray drying is the one of many processes for
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encapsulation of microbial cells to be applied as a starter in fermented food industries. The challenge
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associated with this technique is to produce viable culture, especially with heat sensitive strains
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(Broeckx et al., 2016). Heat applied in spray drying induced the loss of bacterial viability involving
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the combination of protein denaturation, oxidative stress or membrane leakage (Ebrahimi et al, 2018).
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The removal of water during drying may also cause changes in the structure of bacterial membrane
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and detrimental to bacterial cells. The number of viable bacterial cell decreases during drying
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processing. A common method for improving the viability of cultures is the addition of one or more
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protective agents. These agents may be protein (Mohamed et al., 2015) and carbohydrates such as
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glucose, fructose, lactose, mannose, sucrose, sorbitol, adonitol, trehalose, ascorbic acid, skim milk,
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acacia gum, starch and oligosaccharides (Peighambardoust et al., 2011) and mixture of protein and
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carbohydrate (Tantratian et al., 2018). The protective agents prevent the damage of membrane by
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depressing the membrane phase transition. According to the water replacement hypothesis, protective
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agent depress the membrane phase transition by specifically interacting with phospholipid head group.
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The use of carbohydrate as protectants of bacterial cell can alternatively be explained by the water
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replacement hypothesis, which envisages the function of carbohydrate as water substitutes when the
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hydration shell of proteins as well as water molecules around polar residues in membrane
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phospholipids are removed (Ananta et al., 2005).
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The ability to protect bacterial cells of carbohydrate is related to the difference in their glass-
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forming tendencies, which is reflected in their glass transition temperatures (Tg) (Sinha and
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Ranganathan, 1974) and indicated the efficacy of protective agent to protect the bacterial cell during
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drying (Linders et al., 1997). Ananta et al. (2005) reported skim milk, polydextrose and rafilose P95,
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with Tg of 109oC, 108oC and 102oC, respectively, to be used as protectants of Lactobacillus
3
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rhamnosus GG. After drying, the survivals were 65%, 62% and 55%, respectively. However there
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was no study on carbohydrates with different Tg related to protective ability. The limiting amount of carbohydrate in fish meat required the addition of carbohydrate, as an
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ingredient for fermentation of pla-som. The carbohydrate is necessary for lactic acid bacteria to
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produce acidic metabolites and antimicrobial substances. The lactic acid bacteria are able to utilize
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variety of carbohydrates (Hassan et al., 2014; Buron-Moles et al., 2019). Several carbohydrates were
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applied as carbon sources for the fish fermentation, such as sucrose, glucose, lactose, cassava starch
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and steamed rice (Ruangsuwan et al., 2009). Sugars are suitable for carbon sources as they are
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soluble in water while starches and rice have to be cooked prior the bacterial hydrolyzation. The
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amount of carbohydrate ranged from 2-12% is recommended in traditionally Thai fermented fish
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products (Kumar, 2019). Recently, the production of fermented foods based on the application of lactic acid starter
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culture with an industrially important functionality, especial probiotic properties are interested
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(Azhari Ali, 2010). Lactic acid bacteria and their antimicrobial metabolites are widely used in food
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industry in maintaining the quality and safety of food products. A beneficial microorganism to
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control spoilage and render pathogenic activity in foods and fermented foods was emphasized (Singh,
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2018). Fermentation with addition of starter cultures brings benefits including decrease food losses
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and enhance food safety (Talon and Zagorec, 2017). Lactobacillus sp. is known for the ability to
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utilize various type of carbohydrates to produce acids, mainly lactic acid. Lactobacillus plantarum
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FT 35 is an autochthonous culture in Thai sour fermented fish product, called pla-som. It was claimed
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to have ability to produce antagonistic substances against some pathogenic bacteria (Pradeamchai,
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2011).
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The objectives of this study were to find a carbohydrate and optimize concentration that could
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provide a good protection for L. plantarum FT35 during encapsulation and storage. The ability to
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provide as a carbon source for lactic acid bacteria in fermentation will be observed.
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Materials and Methods
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2.1Selection and proper amount of a carbohydrate to be a protective agent of L. plantarum
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2.1.1 Cultivation and preparation of microorganism
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The Lactobacillus plantarum FT 35 (LPFT 35) was isolated from a sour fermented fish
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product, pla-som, and stocked in the Food Microbiology Laboratory of the Food Technology
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Department. It was grown on De Man, Rogosa and Sharpe (MRS) agar and stored in a refrigerator as
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a stock culture. The active culture was prepared by transferring the culture into the 10 ml coconut
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broth medium (Tantratian et al., 2018) and incubated at 37 oC for 24 h, as a seed inoculum. The 2%
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(w/v) culture was transferred into coconut broth 100 ml and incubated at 37 oC for 24 h and applied as
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a starter culture. The working culture was prepared by transferring 2% (v/v) of the starter culture into
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3L of sterile coconut broth medium in a 5L fermenter (B.E. Marubishi, Thailand). The culture was
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incubated at 37 oC with 100 rpm min-1 agitation for 24 h. Cells were harvested by centrifugation
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(7500 rpm for 5 min at 4 oC) and washed once with 0.1 M phosphate buffer pH 6.5-7.0. Cell pellet
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was resuspended in a phosphate buffer solution to reach total solid of 6% (Lian et al., 2002).
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2.1.2 Preparation of protective agents
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Glass transition temperature (Tg) of glucose, sucrose, lactose (Univar, New Zealand),
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maltodextrin DE 10 (Bakeryland, China), and soluble starch (Merck Co., Germany) were determined
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using a Differential Scanning Calorimeter (DSC8000 Model, Perkin Elmer) and shown in Table 1.
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These carbohydrates were added to distilled water to make solutions. All solutions were adjusted to
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achieve the viscosity of 1.6+0.1 Cp. The viscosity of the solutions was determined using a Cannon
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capillary viscometer size 75 A442 at 25 oC (Tantratian et al., 2018). These were applied as protective
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agent solutions in further experiments.
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2.1.3 Spray drying
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Each protective agent solution was mixed with prepared cell suspension. The mixtures were
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adjusted to achieve a cell concentration of 11 log10 CFU mL-1. The mixture was applied as feed-in
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solutions. Spray drying conditions were the inlet temperature of 185 oC, feeding rate of 20 mL min-1
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and outlet temperature of 85+5 oC (Gardiner et al., 2000).
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2.1.4 Determination of the survival and character of the product after spray drying The survival rate was determined by the 0.1 g spray-dried powders were rehydrated with 9.9
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mL sterile peptone water (Merck, Germany) to obtain 1:100 serial dilution (Gardiner et al., 2000) and
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spread on MRS agar, thus incubated at 37 oC for 48 h. Survival rate were calculated as equation (1):
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% Survival = N/N0 × 100 …………………. (1) where N0 represent the number of bacterial before drying and
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N represent the number of bacteria after drying.
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The spray-dried products were scrutinized under Scanning Electron Microscope. The
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Electron Micrographs of the product were determined for the average particle size using Image J for
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windows, version 1.40.
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The number of survived cells was determined by cultivating the proper dilution of dried
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powder products on MRS agar and incubated at 30 oC for 2 days. Determination of the number of
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healthy cells containing in the spray dried products was conducted using the proper dilution of dried
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powder suspension to plate on MRS agar supplemented with 5% NaCl and incubated at 30 oC (Sunny-
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Roberts and Knorr, 2009). The plates were examined after 2-6 days of incubation. The moisture
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content and water activity of the spray dried products were determined following the procedure
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described in the AOAC (2000).
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The carbohydrate protective agent provided the highest survival rate and the highest number
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of healthy cell was selected for further experiments.
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2.1.5 Proper concentration of soluble starch for encapsulation of LPFT35
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The 1.5, 2.5, 3.5, 4.5 and 10% starch solutions were prepared using soluble starch (Merck
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KGaA, Darmstadt, Germany). The cell suspension of LPFT35 was prepared as described. The cell
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suspension was added to the starch solutions to achieve the cell concentration of 1011 CFU mL-1 and
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used for feed solutions. The viscosity of the feed solutions were determined with a cannon capillary
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viscometer at 30 oC (Boza et al., 2004). The spray dry condition was set as described. The survival
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of the culture, healthy cells, moisture and character of the products were determined as described.
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2.1.6 Determination of the survival during storage
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The powdered products were stored in laminate aluminum foil (PP/PE/Alu/PE/PP) bags at
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ambient temperature (28-33 oC) and refrigeration temperature (4+2 oC) for 8 weeks. The number of
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viable cell content in the powdered products was determined by spreading the 1 mL of proper dilution
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on MRS agar. The data were expressed as logarithmic value of survival cells. The moisture content
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(AOAC 2000) and water activity (Aqualab; model series 3, USA) were determined.
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The concentration of selected carbohydrate provided the highest survival rate during
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encapsulation and storage was selected for further experiments.
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2.2 Ability of encapsulated culture in controlling pathogenic bacteria
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An indigenous pla-som fermentation is a spontaneous fermentation. It was prepared by
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addition of 3.0 g table salt, 10.0 g minced garlic and 1.5 g cooked rice to every 100 g of silver barb
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fillet. The controlled fermentation with wet culture was prepared with the same ingredients as
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spontaneous fermentation. The 1 mL of 6 log10 CFU mL-1 of the log phase culture was added. The
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fermentations with LPFT35 culture were prepared with the same ingredient as spontaneous without
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cooked rice and 1 g of spray dried products. The products were kept at several storage time under
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refrigeration temperature (4+2 oC), freshly prepared (less than 1 week old), 1 and 2 month old. Fish
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fillets and other ingredients were kneaded prior to be spiked with pathogenic bacteria. All samples
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were spiked with Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium to about 103
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CFU g-1 each. They were put in plastic bags. The bag was rolled out the air and sealed. It was
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incubated under ambient condition (30-35 oC). Samples were taken at 0, 3 and 5 days for
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microbiological determination, pH and total acidity (expressed as lactic acid). The microbiological
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determination included total aerobic count (AOAC, 2000), E. coli (3M Petrifilm™ E. coli/Coliform
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Count Plates, USA) and Salmonella spp. (AOAC, 2000).
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2.3Statistical analysis
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The experimental design was Completely Randomized Design (CRD) with 95% confidence
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(Cocheran and Cox, 1992). The Duncan’s New Multiple Range Test was applied for comparing the
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mean difference. The experiments were repeated 3 times. The statistical analysis was done using
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SPSS statistical package version 17 (IBM, New York, NY).
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Results and Discussions
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Selection and proper amount of a carbohydrate to be a protective agent of LPFT35
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It was found that protective agents with high Tg provided the higher viable cell number and
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lesser number of injured LPFT35 cells (Figure 1). The spray dried product with glucose had the
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highest moisture content and water activity, 4.51+0.06% and 0.32+0.02, respectively, but had the
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lowest viable cell number and highest cell injury. The lactose, maltodextrin DE 10 and soluble starch,
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which had Tg higher than 100 oC, provided powder products with no significant (p>0.05) difference
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in the number of viable cells, somehow they were significantly (p<0.05) different in the number of
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injured cells. Their products had moisture content of 2.52-2.75% and water activity of 0.21-0.22
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(Table 2). The soluble starch provided the encapsulated product containing high viable cells and
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lowest number of injured cells, 3.5% (Figure 1).
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Polysaccharides, such as maltodextrin DE 10 and soluble starch, formed cross linked with the
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proteins and might interact with head groups of phospholipids and depress membrane phase transition
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(Vereyken et al., 2001). This interaction of polysaccharides with phospholipids greatly depends on
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the flexibility of the structures. On the other hand, the ability of sugar protective agent to stabilize
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cell membrane proteins during drying is due to the ability of the sugar to form hydrogen bonds with
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the protein when water was removed, and prevent protein denaturation by maintaining the protein
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structure (Crown et al., 1996). The ability of the supporting matrix to become amorphous solids
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during drying is beneficial for shielding and separating cells (Wessman, et al., 2013)
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The soluble starch was selected as a protective agent in encapsulation of L. plantarum FT 35
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in further experiments.
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Proper concentration of soluble starch for encapsulation and storage of LPFT35
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The increase of soluble starch content in the feed-in solution caused the viscosity to rise
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(Table 3). This resulted in the production of large particle size powder. The moisture and water
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activity were increased while viability and healthiness of cells were decreased when the starch
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concentration was 3.5% and higher. The feed-in solutions contained 1.5% and 2.5% soluble starch
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showed no significant difference (p>0.05) in number of viable cells contained in the spray dried
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products. The encapsulated products contained viable cells at 94.9+0.12 % and there were
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4.75+0.17% injured. The number of survivors and healthy cells were higher than in those products
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from higher concentration of soluble starch in the feed-in solutions (Figure 2). The number of injured
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cells was increased when the viscosity of feed-in solutions was higher. The data was coincided with
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the report of Tantratian et al. (2018). The higher viscosity of the feed-in solution provided the larger
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droplets after passing through atomizer. These droplets needed longer period of time for dehydration.
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The small droplets had high rate of evaporation during spray drying (Elizondo and Labuza, 1974).
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Lian et al. (2002) reported the decreased in particle size of the spray dried product, the increase in the
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number of surviving cells was found.
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During the storage of the spray-dried products of 2.5% soluble starch, it demonstrated small
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increment in water activity and moisture (Figure 3). The number of viable cells in the spray-dried
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products were more stable than the product with 1.5% soluble starch. This agreed with Champagne et
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al., (1991) who reported the powder products with high moisture content had high in the percentage of
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death during storage. It is recommended that the 4% moisture content was a good-quality parameter
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for dried products. The high in moisture content could cause the oxidation of lipid in the cell
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membrane that resulted in alteration the membrane functions and structures (In’t Veld, et al., 1992).
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Storage at refrigeration temperature (4+2 oC) demonstrated the slower reduction in viable cell
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content than those stored under room condition. Shi et al (2013) suggested that low temperature
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retarded the decrease in viability of the encapsulated cell product.
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The spray dried product of the condition with the 2.5% soluble starch in the feed-in solution was chosen for further experiments.
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Fermentation of pla-som with an encapsulated co-starter Soluble starch has privilege over cassava starch and rice to be applied as a carbon source for
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fermentation. It is soluble and ready to be hydrolyzed by enzymes. The LPFT35 might be an
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amylolytic lactobacillus with ability to produce acidic products. It was reported that the extracellular
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amylase activity was found in several lactobacilli, including L. plantarum (Kim et al., 2008). The
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hydrolyzation resulted in major product of oligosaccharides with degree of polymerization of 3 and 4
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(Gänzle and Follador, 2012). Production of lactic acid from simultaneous saccharification and
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fermentation of cassava starch by L. plantarum was reported (Chookietwattana, 2014).
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The fermentation of pla-som, with a starter powder of LPFT35 added, had acidity increased
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more rapidly than in spontaneous fermentation, no starter added. The pH of starter added
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fermentations reached below 4.5 within 3 day of fermentation (Table 4). The finish product of pla-
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som was suggested to have pH of 4.6 or less (Thai community product standard, 2014). The
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indigenous fermentation of pla-som reached the pH of 4.6 on the fifth day. The addition of
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encapsulated products completed the fermentations faster than spontaneous ones. The freshly dried
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powder demonstrated acid production with no significant different (p>0.05) to the wet active culture,
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while the 4 week old and 8 week old dried powder displayed significantly less (p<0.05). The pH of
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fermented products with addition of a starter were not significantly different (p>0.05) as shown in
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Table 4. The addition of L. casei, about 1010 CFU g-1, to the fermentation of pla-som reduced the pH
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to the level of lower than 4.5 within 3 days (Chaikham and Kaewjinda, 2017).
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At the initial state, the number of bacteria in the spontaneous fermentation was about 3 log10
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CFU g-1. The number of bacteria increased along with fermentation period so as the lactic acid
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bacteria (Table 5). The number of lactic acid bacteria was significantly (p>0.05) higher in
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fermentation with encapsulated LPFT35 added. This confirmed the decreasing in pH faster and
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higher acidity as compared to the control. The number of E. coli, S. Typhimurium and S. aureus were
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rapidly decreased to undetectable within the third day of fermentation of a co-starter added, while the
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spontaneous fermentation demonstrated the undetectable of E. coli, and S. aureus on the fifth day.
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The number of S. Typhimurium was also reduced but still detectable on the fifth day of fermentation
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(Figure 4). Pathogenic bacteria associated with fermented fish are Escherichia coli and Salmonella spp.
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Pumipan and Inmuang (2016) tested fermented fish products in local markets and found that 79.17
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and 25.0 % of products were contained E. coli and Salmonella spp., respectively. The fermentation of
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pla-som with an encapsulated LPFT 35 and soluble starch product was able to eliminate the
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contaminated pathogenic bacteria in the product and completed the fermentation with a shorter period
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of time.
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Conclusion
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Glucose, sucrose, lactose, maltodextrin and soluble starch were applied as protective agents
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for a potential probiotic lactic acid bacteria. The soluble starch, with highest glass transition
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temperature, provided high cell viability and least number of cell injury in the encapsulated powder
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product. The 2.5 percentages of soluble starch in feed-in mixture was suitable to be a heat protectant
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for LPFT35 during spray drying. The encapsulated culture could be kept in a laminated aluminum
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foil bag longer than 2 months under refrigeration temperature.
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The application of the encapsulated products, freshly prepared and stored products, as co-
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starters to the fermentation of pla-som were succeed in rapid reduction of pH. The acidic condition
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with combination of antagonistic effect of LPFT35 suppressed and eliminated the E. coli, S. aureus
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and S. Typhimurium within the 3 days, without a carbon source added. The data indicated soluble
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starch can serve as a good cell protectant during spray drying process and also provide plenty energy
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source for acid production by lactic acid bacteria during fermentation.
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Acknowledgement
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The authors would like to thank the Graduate School of Chulalongkorn University for a Financial
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Support of this study.
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Authors declared no conflict of interest.
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Peighambardoust, S.H., Golshan Tafti, A. and Hesari, J. 2011. Application of spray drying for
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preservation of lactic acid starter cultures: A reviw. Trends in Food Science & Technology
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22(50), 215-224.
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Pradeamchai, M. 2011. Cell protective agents and proper concentration in spray drying of
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Lactobacillus plantarum FT35 for starter culture of fish fermentation. A Master Degree Thesis
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of Science program in Biotechnology, Faculty of Science, Chulalongkorn University.
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Pumipan, T. and Inmaung, U. 2016. Hygiene of Thai traditional fermented fish production in one distric, Khon Kaen Province. KKU Research Journal 16(2), 75-85 Ruangsuwan, C., Brahmawong, C., Pamornsamit, S and Uitragool, P. 2009. A community wisdom
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model: A case study of Pla-som (fermented fish) of Thai-Yor at Tha Kon Yang Villae,
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Kantharawichai Distric, Maha Sarakham Province. Rajabhat Maha Sarakham University Journal
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3(2), 67-78
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Shi, L.E., Li, Z-H, Li, D-T. 2013. Encapsulation of probiotic Lactobacillus bulgaricus in alginate –
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Journal of Food Engineering 117(1):99-104.
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Sinha, R.T. and Ranganathan, D, 1974. A research note: Protective effect of fortified skimmilk is
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suspending medium of freeze drying of difference lactic acid bacteria. Journal of Food Science.
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341 342 343
Singh, V.P. 2018. Recent approach in food bio-preservation – a review. Open Veterinary Journal 8(1): 104–111. doi: 10.4314/ovj.v8i1.16 Sunny-Roberts, E.O. and Knorr, D. 2009. The protective effect of monosodium glutamate on
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survival of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus E-97800 (E800) strains
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during spray-drying and storage in trehalose-containing powders. International Dairy Journal, 19
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Tantratian, S., Wattanaprasert, S. and Suknaisilp, S. 2018. Effect of partial substitution of milk-non-
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fat with xanthan gum for encapsulation of a probiotic Lactobacillus. Journal of Food Processing
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Talon, R. and Zagorec, M. 2017. Special issue: Beneficial microorganisms for food manufacturing-
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fermented and biopreserved foods and beverages. Microorganisms 5(4), 71. doi:
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10.3390/microorganisms5040071
353 354 355
Thai community products standards. 2014. Fermented fish, Pla-som. Thai Industrial Standard Institute, Ministry of Industrial, Bangkok (in Thai). p 1-10. Vereyken, I.J., Chupin, V., Demel, R.A., Smeekens, S.C. and Kruijff, B. 2001. Fructans insert
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Wessman, P., Håkansson, s., Leifer, K. and Rubino, S. 2013. Formulations for freeze-drying of bacteria and their influence on cell survival. Journal of Visualized Experiments 78, 4058
Table 1. Glass transition temperature (Tg) of selected protective agents (Pradeamchai et al., 2012) Protective agent Glucose Sucrose Lactose Maltodextrin DE 10 Soluble starch
Tg (oC) 35.3 75.1 119.3 160.0 241.8
Table 2. Physical characteristics of encapsulated products with various types of carbohydrates Moisture content (%) Water activity (aw) glucose 4.51a+0.06 0.39a+0.01 b sucrose 3.53 +0.04 0.32b+0.02 c lactose 2.75 +0.09 0.22c+0.01 c Maltodextrin DE 10 2.61 +0.05 0.21c+0.02 Soluble starch 2.52c+0.08 0.21c+0.01 Note a,b,c means in the same column with different letter are significantly different (p>0.05); n=3.
Table 3. Some characteristics of feed-in solutions for spray drying process and encapsulated products of Lactobacillus plantarum FT35 Feed in characters encapsulated product characters Soluble starch (%) Viscosity (cP) Particle size* (µm) Moisture (%) aw 1.5 1.088 10.89a + 1.23 2.57a + 0.08 0.21a + 0.01 2.5 1.753 25.63b + 1.08 2.59a + 0.12 0.22a + 0.02 c b 3.5 2.767 37.33 + 1.41 3.01 + 0.02 0.25b + 0.01 d c 4.5 3.809 49.15 + 1.38 3.50 + 0.05 0.27c + 0.01 10.0 9.773 81.22e + 1.44 4.96d + 1.02 0.30d + 0.01 Note; * The average of 30 particles from microscopic images; a, b, c… means in the same column with different letters are significantly (p>0.05) different. n=3.
Table 4. Changes of acidity (%) and pH during fermentation of plasom 0 day 3 day 5 day No starter added 0.20h+0.01 0.55e+0.05 0.95d+0.05 g cd Wet culture 0.25 +0.01 1.10 +0.05 1.70ab+0.06 g c Dry culture 0.25 +0.02 1.20 +0.06 1.80a+0.07 g d 4 week old culture 0.24 +0.02 0.99 +0.04 1.45b+0.06 g d 8 week old culture 0.23 +0.02 0.74 +0.04 1.15cd+0.05 pH No starter added 6.05A+0.20 5.10B+0.18 4.85C+0.18 A CD Wet culture 6.15 +0.25 4.46 +0.21 4.10D+0.17 A D Dry culture 6.15 +0.25 4.15 +0.19 3.95E+0.20 A CD 4 week old culture 6.16 +0.20 4.45 +0.22 4.15D+0.21 8 week old culture 6.04A+0.25 4.81C+0.21 4.45CD+0.22 Note; A,B,….. a, b,…. Means with different letters are significantly (p<0.05) different, n =3. Acidity (%)
Table 5. The changes in total viable count and lactic acid bacteria during fermentation 0 day 3 day 5 day No starter added 3.21d+0.51 6.49d+0.21 8.53d+0.23 a a Wet culture 5.37 +0.14 8.53 +0.20 9.32b+0.12 a a Dry culture 5.42 +0.07 8.42 +0.17 9.61a+0.15 b b 4 week old culture 5.04 +0.12 8.04 +0.05 9.01c+0.07 c c 8 week old culture 4.28 +0.22 7.78 +0.10 8.96c+0.03 Lactic acid No starter added 3.05C+0.13 6.15E+0.16 7.40D+0.44 A B bacteria Wet culture 5.54 +0.07 8.60 +0.14 9.72A+0.22 A A Dry culture 5.67 +0.21 8.81 +0.08 9.93A+0.30 B C 4 week old culture 5.11 +0.08 8.21 +0.03 9.43B+0.05 8 week old culture 4.98B+0.22 8.0D+0.14 9.08C+0.20 Note; A,B,….. a, b,…. Means with different letters are significantly (p<0.05) different, n =3. Total viable count
Figure 1. The viable count and percentage of injured cells of L. plantarum FT35 contained in encapsulated products with various glass transition temperature (Tg) protectants.
Figure 2. Number of viable cells and percentage of injured cells contained in the encapsulated products from various concentrations of soluble starch in feed-in solutions.
(a)
(b)
(c) Figure 3. Changes in viable cell content (a), water activity (b) and moisture (c) during storage of encapsulated products of Lactobacillus plantarum FT35
(a)
(b)
(c) Figure 5. Changes in the number of pathogenic bacteria, E. coli (a) and Salmonella spp. (b) S. aureus (c) during fermentation of pla-som with various type of fermentations; no starter added, wet culture (WC), fresh prepared culture powder (DC), 4 week old culture powder (4wDC), and 8 week old culture powder (8wDC).
Highlight -
The encapsulated product of soluble starch contained the highest viable cells and least injury. The 2.5% soluble starch in feed-in solution for drying is the best condition for cell protection The 8 weeks old encapsulated product was able to be applied as a starter for fermentation. The soluble starch serves as a good carbon source for lactic acid bacteria in the fermentation.
S0023-6438(20)30063-3
DOI:
https://doi.org/10.1016/j.lwt.2020.109075
Reference:
YFSTL 109075
To appear in:
LWT - Food Science and Technology
Received Date: 15 October 2019 Revised Date:
9 December 2019
Accepted Date: 21 January 2020
Please cite this article as: Tantratian, S., Pradeamchai, M., Select a protective agent for encapsulation of Lactobacillus plantarum, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/ j.lwt.2020.109075. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
CRediT author statement Sumate Tantratian: Writing-Reviewing and Editing, Supervision, Conceptualization, Data curation, Visualization Mathurose Pradeamchai: Investigation, Data curation, Formal Analysis
1
Select a protective agent for encapsulation of Lactobacillus plantarum
1
2
Sumate Tantratian1,3*and Mathurose Pradeamchai2
3
1
Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND
4
2
Biotechnology Program, Faculty of Science, Chulalongkorn University, Bangkok, THAILAND
5
3
The development of foods and food additive from innovative microbial fermentation Research Group,
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Faculty of Science, Chulalongkorn University Bangkok 10330, THAILAND
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* corresponding author; [email protected]
8
Abstract
9
Five carbohydrates were applied as protective agents for encapsulation of Lactobacillus
10
plantarum FT 35 which produced high acid and had ability to inhibit some pathogenic bacteria. They
11
had glass transition temperatures in ascending order from glucose, sucrose, lactose, maltodextrin to
12
soluble starch. The soluble starch demonstrated the best protection to cell during drying process and
13
storage. The accretion of soluble starch higher than 2.5% in feed-in mixture for spray drying
14
exhibited the increase in the viscosity of mixtures and the particle size of products. These products
15
contained less number of viable cells and higher cell injury. The encapsulated products with 2.5%
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soluble starch exhibited a better storage stability than those with 1.5%. The encapsulated product was
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applied as a co-starter in pla-som fermentation without additional of carbon sources and also
18
demonstrated fast acid production. The fermentation with encapsulated products displayed better
19
inhibitory effect on inoculated pathogenic bacteria than indigenous fermentation. The soluble starch
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serves as a protective agent for encapsulated culture and also a good carbon source for lactic acid
21
bacteria in the fermentation of pla-som.
22
Keywords; glass transition temperature, carbohydrate, encapsulation, carbon source, pla-som
23
fermentation
24
2
25
Introduction
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Encapsulation is the process to protect bacterial cell or bioactive substance against harsh
27
environment in wall materials (Arpagaus et al., 2018). It allow probiotics to be widely applied in
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fresh or dried food markets (Martín et al., 2015). Spray drying is the one of many processes for
29
encapsulation of microbial cells to be applied as a starter in fermented food industries. The challenge
30
associated with this technique is to produce viable culture, especially with heat sensitive strains
31
(Broeckx et al., 2016). Heat applied in spray drying induced the loss of bacterial viability involving
32
the combination of protein denaturation, oxidative stress or membrane leakage (Ebrahimi et al, 2018).
33
The removal of water during drying may also cause changes in the structure of bacterial membrane
34
and detrimental to bacterial cells. The number of viable bacterial cell decreases during drying
35
processing. A common method for improving the viability of cultures is the addition of one or more
36
protective agents. These agents may be protein (Mohamed et al., 2015) and carbohydrates such as
37
glucose, fructose, lactose, mannose, sucrose, sorbitol, adonitol, trehalose, ascorbic acid, skim milk,
38
acacia gum, starch and oligosaccharides (Peighambardoust et al., 2011) and mixture of protein and
39
carbohydrate (Tantratian et al., 2018). The protective agents prevent the damage of membrane by
40
depressing the membrane phase transition. According to the water replacement hypothesis, protective
41
agent depress the membrane phase transition by specifically interacting with phospholipid head group.
42
The use of carbohydrate as protectants of bacterial cell can alternatively be explained by the water
43
replacement hypothesis, which envisages the function of carbohydrate as water substitutes when the
44
hydration shell of proteins as well as water molecules around polar residues in membrane
45
phospholipids are removed (Ananta et al., 2005).
46
The ability to protect bacterial cells of carbohydrate is related to the difference in their glass-
47
forming tendencies, which is reflected in their glass transition temperatures (Tg) (Sinha and
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Ranganathan, 1974) and indicated the efficacy of protective agent to protect the bacterial cell during
49
drying (Linders et al., 1997). Ananta et al. (2005) reported skim milk, polydextrose and rafilose P95,
50
with Tg of 109oC, 108oC and 102oC, respectively, to be used as protectants of Lactobacillus
3
51
rhamnosus GG. After drying, the survivals were 65%, 62% and 55%, respectively. However there
52
was no study on carbohydrates with different Tg related to protective ability. The limiting amount of carbohydrate in fish meat required the addition of carbohydrate, as an
53 54
ingredient for fermentation of pla-som. The carbohydrate is necessary for lactic acid bacteria to
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produce acidic metabolites and antimicrobial substances. The lactic acid bacteria are able to utilize
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variety of carbohydrates (Hassan et al., 2014; Buron-Moles et al., 2019). Several carbohydrates were
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applied as carbon sources for the fish fermentation, such as sucrose, glucose, lactose, cassava starch
58
and steamed rice (Ruangsuwan et al., 2009). Sugars are suitable for carbon sources as they are
59
soluble in water while starches and rice have to be cooked prior the bacterial hydrolyzation. The
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amount of carbohydrate ranged from 2-12% is recommended in traditionally Thai fermented fish
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products (Kumar, 2019). Recently, the production of fermented foods based on the application of lactic acid starter
62 63
culture with an industrially important functionality, especial probiotic properties are interested
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(Azhari Ali, 2010). Lactic acid bacteria and their antimicrobial metabolites are widely used in food
65
industry in maintaining the quality and safety of food products. A beneficial microorganism to
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control spoilage and render pathogenic activity in foods and fermented foods was emphasized (Singh,
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2018). Fermentation with addition of starter cultures brings benefits including decrease food losses
68
and enhance food safety (Talon and Zagorec, 2017). Lactobacillus sp. is known for the ability to
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utilize various type of carbohydrates to produce acids, mainly lactic acid. Lactobacillus plantarum
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FT 35 is an autochthonous culture in Thai sour fermented fish product, called pla-som. It was claimed
71
to have ability to produce antagonistic substances against some pathogenic bacteria (Pradeamchai,
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2011).
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The objectives of this study were to find a carbohydrate and optimize concentration that could
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provide a good protection for L. plantarum FT35 during encapsulation and storage. The ability to
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provide as a carbon source for lactic acid bacteria in fermentation will be observed.
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Materials and Methods
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2.1Selection and proper amount of a carbohydrate to be a protective agent of L. plantarum
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2.1.1 Cultivation and preparation of microorganism
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The Lactobacillus plantarum FT 35 (LPFT 35) was isolated from a sour fermented fish
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product, pla-som, and stocked in the Food Microbiology Laboratory of the Food Technology
81
Department. It was grown on De Man, Rogosa and Sharpe (MRS) agar and stored in a refrigerator as
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a stock culture. The active culture was prepared by transferring the culture into the 10 ml coconut
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broth medium (Tantratian et al., 2018) and incubated at 37 oC for 24 h, as a seed inoculum. The 2%
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(w/v) culture was transferred into coconut broth 100 ml and incubated at 37 oC for 24 h and applied as
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a starter culture. The working culture was prepared by transferring 2% (v/v) of the starter culture into
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3L of sterile coconut broth medium in a 5L fermenter (B.E. Marubishi, Thailand). The culture was
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incubated at 37 oC with 100 rpm min-1 agitation for 24 h. Cells were harvested by centrifugation
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(7500 rpm for 5 min at 4 oC) and washed once with 0.1 M phosphate buffer pH 6.5-7.0. Cell pellet
89
was resuspended in a phosphate buffer solution to reach total solid of 6% (Lian et al., 2002).
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2.1.2 Preparation of protective agents
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Glass transition temperature (Tg) of glucose, sucrose, lactose (Univar, New Zealand),
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maltodextrin DE 10 (Bakeryland, China), and soluble starch (Merck Co., Germany) were determined
93
using a Differential Scanning Calorimeter (DSC8000 Model, Perkin Elmer) and shown in Table 1.
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These carbohydrates were added to distilled water to make solutions. All solutions were adjusted to
95
achieve the viscosity of 1.6+0.1 Cp. The viscosity of the solutions was determined using a Cannon
96
capillary viscometer size 75 A442 at 25 oC (Tantratian et al., 2018). These were applied as protective
97
agent solutions in further experiments.
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2.1.3 Spray drying
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Each protective agent solution was mixed with prepared cell suspension. The mixtures were
100
adjusted to achieve a cell concentration of 11 log10 CFU mL-1. The mixture was applied as feed-in
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solutions. Spray drying conditions were the inlet temperature of 185 oC, feeding rate of 20 mL min-1
102
and outlet temperature of 85+5 oC (Gardiner et al., 2000).
5
103 104
2.1.4 Determination of the survival and character of the product after spray drying The survival rate was determined by the 0.1 g spray-dried powders were rehydrated with 9.9
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mL sterile peptone water (Merck, Germany) to obtain 1:100 serial dilution (Gardiner et al., 2000) and
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spread on MRS agar, thus incubated at 37 oC for 48 h. Survival rate were calculated as equation (1):
107 108
% Survival = N/N0 × 100 …………………. (1) where N0 represent the number of bacterial before drying and
109
N represent the number of bacteria after drying.
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The spray-dried products were scrutinized under Scanning Electron Microscope. The
111
Electron Micrographs of the product were determined for the average particle size using Image J for
112
windows, version 1.40.
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The number of survived cells was determined by cultivating the proper dilution of dried
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powder products on MRS agar and incubated at 30 oC for 2 days. Determination of the number of
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healthy cells containing in the spray dried products was conducted using the proper dilution of dried
116
powder suspension to plate on MRS agar supplemented with 5% NaCl and incubated at 30 oC (Sunny-
117
Roberts and Knorr, 2009). The plates were examined after 2-6 days of incubation. The moisture
118
content and water activity of the spray dried products were determined following the procedure
119
described in the AOAC (2000).
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The carbohydrate protective agent provided the highest survival rate and the highest number
121
of healthy cell was selected for further experiments.
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2.1.5 Proper concentration of soluble starch for encapsulation of LPFT35
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The 1.5, 2.5, 3.5, 4.5 and 10% starch solutions were prepared using soluble starch (Merck
124
KGaA, Darmstadt, Germany). The cell suspension of LPFT35 was prepared as described. The cell
125
suspension was added to the starch solutions to achieve the cell concentration of 1011 CFU mL-1 and
126
used for feed solutions. The viscosity of the feed solutions were determined with a cannon capillary
6
127
viscometer at 30 oC (Boza et al., 2004). The spray dry condition was set as described. The survival
128
of the culture, healthy cells, moisture and character of the products were determined as described.
129
2.1.6 Determination of the survival during storage
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The powdered products were stored in laminate aluminum foil (PP/PE/Alu/PE/PP) bags at
131
ambient temperature (28-33 oC) and refrigeration temperature (4+2 oC) for 8 weeks. The number of
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viable cell content in the powdered products was determined by spreading the 1 mL of proper dilution
133
on MRS agar. The data were expressed as logarithmic value of survival cells. The moisture content
134
(AOAC 2000) and water activity (Aqualab; model series 3, USA) were determined.
135
The concentration of selected carbohydrate provided the highest survival rate during
136
encapsulation and storage was selected for further experiments.
137
2.2 Ability of encapsulated culture in controlling pathogenic bacteria
138
An indigenous pla-som fermentation is a spontaneous fermentation. It was prepared by
139
addition of 3.0 g table salt, 10.0 g minced garlic and 1.5 g cooked rice to every 100 g of silver barb
140
fillet. The controlled fermentation with wet culture was prepared with the same ingredients as
141
spontaneous fermentation. The 1 mL of 6 log10 CFU mL-1 of the log phase culture was added. The
142
fermentations with LPFT35 culture were prepared with the same ingredient as spontaneous without
143
cooked rice and 1 g of spray dried products. The products were kept at several storage time under
144
refrigeration temperature (4+2 oC), freshly prepared (less than 1 week old), 1 and 2 month old. Fish
145
fillets and other ingredients were kneaded prior to be spiked with pathogenic bacteria. All samples
146
were spiked with Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium to about 103
147
CFU g-1 each. They were put in plastic bags. The bag was rolled out the air and sealed. It was
148
incubated under ambient condition (30-35 oC). Samples were taken at 0, 3 and 5 days for
149
microbiological determination, pH and total acidity (expressed as lactic acid). The microbiological
150
determination included total aerobic count (AOAC, 2000), E. coli (3M Petrifilm™ E. coli/Coliform
151
Count Plates, USA) and Salmonella spp. (AOAC, 2000).
152
2.3Statistical analysis
7
153
The experimental design was Completely Randomized Design (CRD) with 95% confidence
154
(Cocheran and Cox, 1992). The Duncan’s New Multiple Range Test was applied for comparing the
155
mean difference. The experiments were repeated 3 times. The statistical analysis was done using
156
SPSS statistical package version 17 (IBM, New York, NY).
157
Results and Discussions
158
Selection and proper amount of a carbohydrate to be a protective agent of LPFT35
159
It was found that protective agents with high Tg provided the higher viable cell number and
160
lesser number of injured LPFT35 cells (Figure 1). The spray dried product with glucose had the
161
highest moisture content and water activity, 4.51+0.06% and 0.32+0.02, respectively, but had the
162
lowest viable cell number and highest cell injury. The lactose, maltodextrin DE 10 and soluble starch,
163
which had Tg higher than 100 oC, provided powder products with no significant (p>0.05) difference
164
in the number of viable cells, somehow they were significantly (p<0.05) different in the number of
165
injured cells. Their products had moisture content of 2.52-2.75% and water activity of 0.21-0.22
166
(Table 2). The soluble starch provided the encapsulated product containing high viable cells and
167
lowest number of injured cells, 3.5% (Figure 1).
168
Polysaccharides, such as maltodextrin DE 10 and soluble starch, formed cross linked with the
169
proteins and might interact with head groups of phospholipids and depress membrane phase transition
170
(Vereyken et al., 2001). This interaction of polysaccharides with phospholipids greatly depends on
171
the flexibility of the structures. On the other hand, the ability of sugar protective agent to stabilize
172
cell membrane proteins during drying is due to the ability of the sugar to form hydrogen bonds with
173
the protein when water was removed, and prevent protein denaturation by maintaining the protein
174
structure (Crown et al., 1996). The ability of the supporting matrix to become amorphous solids
175
during drying is beneficial for shielding and separating cells (Wessman, et al., 2013)
176
The soluble starch was selected as a protective agent in encapsulation of L. plantarum FT 35
177
in further experiments.
178
Proper concentration of soluble starch for encapsulation and storage of LPFT35
8
179
The increase of soluble starch content in the feed-in solution caused the viscosity to rise
180
(Table 3). This resulted in the production of large particle size powder. The moisture and water
181
activity were increased while viability and healthiness of cells were decreased when the starch
182
concentration was 3.5% and higher. The feed-in solutions contained 1.5% and 2.5% soluble starch
183
showed no significant difference (p>0.05) in number of viable cells contained in the spray dried
184
products. The encapsulated products contained viable cells at 94.9+0.12 % and there were
185
4.75+0.17% injured. The number of survivors and healthy cells were higher than in those products
186
from higher concentration of soluble starch in the feed-in solutions (Figure 2). The number of injured
187
cells was increased when the viscosity of feed-in solutions was higher. The data was coincided with
188
the report of Tantratian et al. (2018). The higher viscosity of the feed-in solution provided the larger
189
droplets after passing through atomizer. These droplets needed longer period of time for dehydration.
190
The small droplets had high rate of evaporation during spray drying (Elizondo and Labuza, 1974).
191
Lian et al. (2002) reported the decreased in particle size of the spray dried product, the increase in the
192
number of surviving cells was found.
193
During the storage of the spray-dried products of 2.5% soluble starch, it demonstrated small
194
increment in water activity and moisture (Figure 3). The number of viable cells in the spray-dried
195
products were more stable than the product with 1.5% soluble starch. This agreed with Champagne et
196
al., (1991) who reported the powder products with high moisture content had high in the percentage of
197
death during storage. It is recommended that the 4% moisture content was a good-quality parameter
198
for dried products. The high in moisture content could cause the oxidation of lipid in the cell
199
membrane that resulted in alteration the membrane functions and structures (In’t Veld, et al., 1992).
200
Storage at refrigeration temperature (4+2 oC) demonstrated the slower reduction in viable cell
201
content than those stored under room condition. Shi et al (2013) suggested that low temperature
202
retarded the decrease in viability of the encapsulated cell product.
203 204
The spray dried product of the condition with the 2.5% soluble starch in the feed-in solution was chosen for further experiments.
9
205 206 207
Fermentation of pla-som with an encapsulated co-starter Soluble starch has privilege over cassava starch and rice to be applied as a carbon source for
208
fermentation. It is soluble and ready to be hydrolyzed by enzymes. The LPFT35 might be an
209
amylolytic lactobacillus with ability to produce acidic products. It was reported that the extracellular
210
amylase activity was found in several lactobacilli, including L. plantarum (Kim et al., 2008). The
211
hydrolyzation resulted in major product of oligosaccharides with degree of polymerization of 3 and 4
212
(Gänzle and Follador, 2012). Production of lactic acid from simultaneous saccharification and
213
fermentation of cassava starch by L. plantarum was reported (Chookietwattana, 2014).
214
The fermentation of pla-som, with a starter powder of LPFT35 added, had acidity increased
215
more rapidly than in spontaneous fermentation, no starter added. The pH of starter added
216
fermentations reached below 4.5 within 3 day of fermentation (Table 4). The finish product of pla-
217
som was suggested to have pH of 4.6 or less (Thai community product standard, 2014). The
218
indigenous fermentation of pla-som reached the pH of 4.6 on the fifth day. The addition of
219
encapsulated products completed the fermentations faster than spontaneous ones. The freshly dried
220
powder demonstrated acid production with no significant different (p>0.05) to the wet active culture,
221
while the 4 week old and 8 week old dried powder displayed significantly less (p<0.05). The pH of
222
fermented products with addition of a starter were not significantly different (p>0.05) as shown in
223
Table 4. The addition of L. casei, about 1010 CFU g-1, to the fermentation of pla-som reduced the pH
224
to the level of lower than 4.5 within 3 days (Chaikham and Kaewjinda, 2017).
225
At the initial state, the number of bacteria in the spontaneous fermentation was about 3 log10
226
CFU g-1. The number of bacteria increased along with fermentation period so as the lactic acid
227
bacteria (Table 5). The number of lactic acid bacteria was significantly (p>0.05) higher in
228
fermentation with encapsulated LPFT35 added. This confirmed the decreasing in pH faster and
229
higher acidity as compared to the control. The number of E. coli, S. Typhimurium and S. aureus were
230
rapidly decreased to undetectable within the third day of fermentation of a co-starter added, while the
10
231
spontaneous fermentation demonstrated the undetectable of E. coli, and S. aureus on the fifth day.
232
The number of S. Typhimurium was also reduced but still detectable on the fifth day of fermentation
233
(Figure 4). Pathogenic bacteria associated with fermented fish are Escherichia coli and Salmonella spp.
234 235
Pumipan and Inmuang (2016) tested fermented fish products in local markets and found that 79.17
236
and 25.0 % of products were contained E. coli and Salmonella spp., respectively. The fermentation of
237
pla-som with an encapsulated LPFT 35 and soluble starch product was able to eliminate the
238
contaminated pathogenic bacteria in the product and completed the fermentation with a shorter period
239
of time.
240
Conclusion
241
Glucose, sucrose, lactose, maltodextrin and soluble starch were applied as protective agents
242
for a potential probiotic lactic acid bacteria. The soluble starch, with highest glass transition
243
temperature, provided high cell viability and least number of cell injury in the encapsulated powder
244
product. The 2.5 percentages of soluble starch in feed-in mixture was suitable to be a heat protectant
245
for LPFT35 during spray drying. The encapsulated culture could be kept in a laminated aluminum
246
foil bag longer than 2 months under refrigeration temperature.
247
The application of the encapsulated products, freshly prepared and stored products, as co-
248
starters to the fermentation of pla-som were succeed in rapid reduction of pH. The acidic condition
249
with combination of antagonistic effect of LPFT35 suppressed and eliminated the E. coli, S. aureus
250
and S. Typhimurium within the 3 days, without a carbon source added. The data indicated soluble
251
starch can serve as a good cell protectant during spray drying process and also provide plenty energy
252
source for acid production by lactic acid bacteria during fermentation.
253
Acknowledgement
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The authors would like to thank the Graduate School of Chulalongkorn University for a Financial
255
Support of this study.
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256
Authors declared no conflict of interest.
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Table 1. Glass transition temperature (Tg) of selected protective agents (Pradeamchai et al., 2012) Protective agent Glucose Sucrose Lactose Maltodextrin DE 10 Soluble starch
Tg (oC) 35.3 75.1 119.3 160.0 241.8
Table 2. Physical characteristics of encapsulated products with various types of carbohydrates Moisture content (%) Water activity (aw) glucose 4.51a+0.06 0.39a+0.01 b sucrose 3.53 +0.04 0.32b+0.02 c lactose 2.75 +0.09 0.22c+0.01 c Maltodextrin DE 10 2.61 +0.05 0.21c+0.02 Soluble starch 2.52c+0.08 0.21c+0.01 Note a,b,c means in the same column with different letter are significantly different (p>0.05); n=3.
Table 3. Some characteristics of feed-in solutions for spray drying process and encapsulated products of Lactobacillus plantarum FT35 Feed in characters encapsulated product characters Soluble starch (%) Viscosity (cP) Particle size* (µm) Moisture (%) aw 1.5 1.088 10.89a + 1.23 2.57a + 0.08 0.21a + 0.01 2.5 1.753 25.63b + 1.08 2.59a + 0.12 0.22a + 0.02 c b 3.5 2.767 37.33 + 1.41 3.01 + 0.02 0.25b + 0.01 d c 4.5 3.809 49.15 + 1.38 3.50 + 0.05 0.27c + 0.01 10.0 9.773 81.22e + 1.44 4.96d + 1.02 0.30d + 0.01 Note; * The average of 30 particles from microscopic images; a, b, c… means in the same column with different letters are significantly (p>0.05) different. n=3.
Table 4. Changes of acidity (%) and pH during fermentation of plasom 0 day 3 day 5 day No starter added 0.20h+0.01 0.55e+0.05 0.95d+0.05 g cd Wet culture 0.25 +0.01 1.10 +0.05 1.70ab+0.06 g c Dry culture 0.25 +0.02 1.20 +0.06 1.80a+0.07 g d 4 week old culture 0.24 +0.02 0.99 +0.04 1.45b+0.06 g d 8 week old culture 0.23 +0.02 0.74 +0.04 1.15cd+0.05 pH No starter added 6.05A+0.20 5.10B+0.18 4.85C+0.18 A CD Wet culture 6.15 +0.25 4.46 +0.21 4.10D+0.17 A D Dry culture 6.15 +0.25 4.15 +0.19 3.95E+0.20 A CD 4 week old culture 6.16 +0.20 4.45 +0.22 4.15D+0.21 8 week old culture 6.04A+0.25 4.81C+0.21 4.45CD+0.22 Note; A,B,….. a, b,…. Means with different letters are significantly (p<0.05) different, n =3. Acidity (%)
Table 5. The changes in total viable count and lactic acid bacteria during fermentation 0 day 3 day 5 day No starter added 3.21d+0.51 6.49d+0.21 8.53d+0.23 a a Wet culture 5.37 +0.14 8.53 +0.20 9.32b+0.12 a a Dry culture 5.42 +0.07 8.42 +0.17 9.61a+0.15 b b 4 week old culture 5.04 +0.12 8.04 +0.05 9.01c+0.07 c c 8 week old culture 4.28 +0.22 7.78 +0.10 8.96c+0.03 Lactic acid No starter added 3.05C+0.13 6.15E+0.16 7.40D+0.44 A B bacteria Wet culture 5.54 +0.07 8.60 +0.14 9.72A+0.22 A A Dry culture 5.67 +0.21 8.81 +0.08 9.93A+0.30 B C 4 week old culture 5.11 +0.08 8.21 +0.03 9.43B+0.05 8 week old culture 4.98B+0.22 8.0D+0.14 9.08C+0.20 Note; A,B,….. a, b,…. Means with different letters are significantly (p<0.05) different, n =3. Total viable count
Figure 1. The viable count and percentage of injured cells of L. plantarum FT35 contained in encapsulated products with various glass transition temperature (Tg) protectants.
Figure 2. Number of viable cells and percentage of injured cells contained in the encapsulated products from various concentrations of soluble starch in feed-in solutions.
(a)
(b)
(c) Figure 3. Changes in viable cell content (a), water activity (b) and moisture (c) during storage of encapsulated products of Lactobacillus plantarum FT35
(a)
(b)
(c) Figure 5. Changes in the number of pathogenic bacteria, E. coli (a) and Salmonella spp. (b) S. aureus (c) during fermentation of pla-som with various type of fermentations; no starter added, wet culture (WC), fresh prepared culture powder (DC), 4 week old culture powder (4wDC), and 8 week old culture powder (8wDC).
Highlight -
The encapsulated product of soluble starch contained the highest viable cells and least injury. The 2.5% soluble starch in feed-in solution for drying is the best condition for cell protection The 8 weeks old encapsulated product was able to be applied as a starter for fermentation. The soluble starch serves as a good carbon source for lactic acid bacteria in the fermentation.