Enhancing the Viability of Lactobacillus plantarum as Probiotics

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Food and Beverage Chemistry/Biochemistry

Enhancing the Viability of Lactobacillus plantarum as Probiotics through Encapsulation with the High Internal Phase Emulsions Stabilized with Whey Protein Isolate Microgels Jiuling Su, Xiaoqi Wang, Wei Li, Ligen Chen, Xiaoxiong Zeng, Qingrong Huang, and Bing Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03807 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Journal of Agricultural and Food Chemistry

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Enhancing the Viability of Lactobacillus plantarum as Probiotics through

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Encapsulation with the High Internal Phase Emulsions Stabilized with Whey

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Protein Isolate Microgels

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Jiuling Su†#, Xiaoqi Wang‡#, Wei Li†, Ligen Chen‖, Xiaoxiong Zeng†, Qingrong

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Huang‡, Bing Hu†*

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† College

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Weigang, Nanjing, Jiangsu, 210095, P. R. China.

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‡

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New Jersey 08901, USA.

of Food Science and Technology, Nanjing Agricultural University, 1

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick,

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‖Department

of Bioengineering, School of Marine and Bioengineering, Yancheng

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Institute of Technology, Yancheng , China.

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*To whom correspondence should be addressed.

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Email: [email protected]

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# Contribute

equally

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ACS Paragon Plus Environment


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ABSTRACT Probiotics with positive physiological effects on intestinal microflora

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populations of the host are popular in functional foods. Low relative humidity (RH)

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and temperature are beneficial for probiotics survival. In the present study, freeze-

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dried Lactobacillus plantarum powder, one representative of probiotics was

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encapsulated in the high internal phase emulsions (HIPEs) stabilized with whey

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protein isolate (WPI) microgels, in order to avoid the contact of water.

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Homogeneously dispersed WPI microgels with particle sizes around 300 nm were

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formed through thermal treatment of WPI solution. The particle size of the microgels

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decreased with the elevation of protein concentrations as well as the leaving away of

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pH values from the isoelectric point of the protein. When internal oil phase volume

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fractions were higher than 80% (w/w), WPI microgels with concentrations higher than

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4.0 wt%, prepared at pH 4.0, pH 6.0 and pH 7.0 conditions could stabilize the oil to

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form homogenous HIPEs with tilting stability. The HIPEs thus formed had a cellular

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and tunable pore structure that could resist mechanical perturbation. Encapsulation of

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Lactobacillus plantarum within HIPEs successfully increased the cell viability after

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pasteurization processing. And the protective effect was even improved with the

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elevation of oil volume fraction and increase of WPI microgel concentrations. Under

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different pH conditions, the strongest protective effect appeared at pH 4.0, when the

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WPI microgels accumulated on the oil droplet surface. By combining the large

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amount of oil and the protein microgel layer on the oil-water interface, as two

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specialties, the HIPEs were demonstrated to have high potential for enhancing the

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viability of probiotics during food thermal processing.

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INTRODUCTION

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Probiotics are viable microorganisms and substances proven to have health-

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promoting effects on humans and animals, especially with positive physiological

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bioactivities on intestinal microflora populations of the host1. Common probiotics

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include

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Lactobacillus acidophilus and Lactobacillus casei, as well as yeasts, for instance,

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Saccharomycces cerevisiae. Lactic acid bacteria are a group of microorganisms in the

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gastrointestinal tract of humans and animals, which have been verified to improve

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intestinal tract health, enhance body immunity, reduce cholesterol content, inhibit

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harmful microorganisms and so on2-4. Thereinto, a widespread heterofermentative

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bacterium, Lactobacillus plantarum, is ubiquitous in milk products, meat, certain

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cheeses, as well as fermented vegetables, sausages and stockfish. The high contents of

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this microorganism in foods make it an ideal candidate for the development of

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probiotics.

Bifidobacterium,

Lactobacillus,

such

as

Lactobacillus

salivarius,

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However, several challenges limit the application of probiotics as supplements in

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foods, such as the poor viability during processing and prolonged storage, and the low

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survival rate in acidic conditions and digestive fluids especially the bile5. Relative

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humidity (RH) and temperature are known to be critical in determining probiotic

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viability during food processing and ambient storage. Low RH and temperature are

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beneficial for the cell survival6-8.

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Efforts have been made to elevate the survival rate of probiotics in adverse

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environments, among which encapsulation shows high potential in real applications.

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Many approaches, such as spray drying, emulsification and freeze-drying have been

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utilized to improve the encapsulation quality. However, even though various drying

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strategies are widely adopted in long-term storage of probiotics, the biggest problem

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in applying the dried probiotics in food processing is still not addressed yet.

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Specifically, food systems are commonly within aqueous environment. When dried

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probiotics are added in, water can permeate into the dried capsules at extremely fast

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rate, and subsequently lead to the inactivation or even death of the probiotics.

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Emulsions are propitious to avoid the humid environment during food processing, due

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to their oil-in-water (O/W) droplet microstructures that can accommodate the

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probiotics in the oil phase. When the internal oil phase fraction is higher than 74%,

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normal emulsions transform to the unique high internal phase emulsions (HIPEs)9,

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which are broadly applied in drug delivery, cosmetic formulations, porous polymer

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materials, bioactive substance protection, etc10-13. Due to the extremely high internal

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oil phase fraction and the low water activity, HIPEs exhibit the superiority of

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increasing the loading contents of probiotics as well as restricting the contact of water.

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Besides, stabilization of the oil droplet interfaces with emulsifiers and the enhanced

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viscoelastic properties of the adsorbed polymer layer account mainly for the

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formation mechanism of the HIPEs.

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Interfacial stabilizers play a pivotal role in the preparation of HIPEs.

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Conventionally, large amounts of surfactants are demanded during HIPEs

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manufacture14,15. Nevertheless, evidence showed that vast surfactants are detrimental

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to human health16,17. Biopolymers have recently shown potential in stabilizing

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HIPEs18. Microgels are the crosslinked polymer networks with the nanoscale sizes

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and gel-like features. In addition, microgels possess both polymeric and particle-like

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characteristics19, which can adsorb onto the oil/water interface and stabilize the

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emulsion systems20. Whey protein isolate (WPI) is a by-product generated during the

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cheese production, which has numerous nutritional and functional properties such as

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antiviral, anti-aging, anti-cancer, emulsification and gel-forming properties21-23.

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Covalently cross-linked WPI microgels can be prepared through complex interplay of

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heat denaturation, aggregation, electrostatic repulsion, and disulfide bonds

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formation24,25. It has been demonstrated that the HIPEs prepared with WPI microgels

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as surfactant generated substantially higher stability than the HIPEs prepared using

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similar loadings of non-gelled WPI26.

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In the present study, HIPEs stabilized with the heating-induced WPI microgels

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were applied to encapsulate probiotics, and Lactobacillus plantarum was selected as

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one representative of the probiotics. Lactobacillus plantarum was firstly freeze-dried

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with skim milk, trehalose and sodium acetate, then dispersed in edible oil, serving as

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the internal phase of the HIPEs. The effects of different WPI concentrations and

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environmental pH conditions on the characteristics of WPI microgels were

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investigated. Subsequently, the influence of microgels concentrations and pH

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conditions in aqueous phase, as well as the volume fractions of oil phase were studied

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towards the physical properties of the formed HIPEs. Furthermore, the viability of

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heat-treated Lactobacillus plantarum encapsulated in HIPEs formed using different

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above-mentioned preparation variables were discussed as well.

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MATERIALS AND METHODS

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Materials. biPro WPI with purity about 90.9% was obtained from Davisco

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Foods International Inc. (America). The WPI contains approximately 50% of β-

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lactoglobulin and 10% of immunoglobulins, which are considered as globular

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proteins. Grape seed oil was obtained from OLITALIA Co., Ltd (Italy), Nile Red was

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obtained from Shanghai Ryon Biological Technology Co., Ltd (China), and

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Lactobacillus plantarum was purchased from China General Microbiological Culture

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Collection Center (Beijing, China). Sodium chloride, sodium azide, hydrochloric acid,

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sodium hydroxide, phosphotungstic acid, trehalose and sodium acetate were obtained

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from Sinopharm Chemical Reagent Co., Ltd (China). Skim milk, agar and De Man,

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Rogosa and Sharpe (MRS) broth were obtained from Beijing Solarbio Science &

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Technology Co., Ltd (China).

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Preparation of WPI Microgels. First, 4.0 wt% WPI powder was dissolved in

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distilled water with 160 mg/L Na+. The solutions were then adjusted to different pH

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environments of pH 3.0, pH 4.0, pH 6.0 and pH 7.0, followed by stirring for 2 h at 25

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0C,

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WPI solutions were heated at 85 0C for 15 min, and subsequently homogenized by a

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D-3L high pressure homogenization (PhD-Tech) at 50000 psi for 3 cycles, followed

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by cooling to 4 0C, to obtain the WPI microgels.

and stored at 4 0C overnight to obtain the final WPI solutions. Afterwards, the

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The Particle Size and ζ-Potential of the Whey Protein Isolate Microgels. The

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particle size and ζ-potential of the whey protein microgels were characterized by

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using a Malvern ZS90 dynamic light scattering (DLS) system (Malvern Instruments

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Ltd, UK) at 25 0C. Each sample with whey protein concentrations of 0.5 wt%, 1.0

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wt%, 4.0 wt% and 10.0 wt% with respect to the aqueous phase, and pH values of the

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protein aqueous solution at pH 3.0, pH 4.0, pH 6.0 and pH 7.0 was measured for at

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least three times. The measurements were conducted at a fixed angle of 900 as

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indicated by the equipment. All the samples for the DLS measurements were in the

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same protein concentration of 0.1 wt% (1 mg/mL) and were in the same measurement

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condition.

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Morphology of the WPI Microgels. The morphology of the WPI microgels was

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investigated by a transmission electron microscope (TEM) machine (JEM-1400plus,

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JEOL Ltd., Japan) using the previously reported method with minor modification27.

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One droplet of WPI microgels with suitable dilution ratio was placed on a carbon

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support film mounted on a copper grid and the droplet was wiped away from the edge

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with a piece of filter paper and air dried. One droplet of 2% phosphotungstic acid was

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added for staining purpose. The fully dried samples were then placed in TEM for

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imaging. The accelerating voltage used was 100 kV.

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Preparation of Freeze-dried Lactobacillus plantarum. Stock Lactobacillus

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plantarum culture was inoculated in MRS broth and incubated at 30 0C for 16 h. Cell

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pellets were collected by centrifugation at 4000 rpm for 3 min at 4 0C. The pellets

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were washed with sterile normal saline for three times and then suspended in ten-fold

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volume of freeze-dried protectant, containing 15.5 wt% dried skim milk, 4.0 wt%

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trehalose and 0.2 wt% sodium acetate. The suspension was freeze-dried using a

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LyoQuest freezing dryer (Telstar, Spain) at -55 0C for 30 h after pre-freezing at -20 0C

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for 24 h. The viable Lactobacillus plantarum counts of the freeze-dried powder can

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reach a population of 109 CFU/g. The freeze-dried powder was stored at -20 0C before

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further experiments.

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Encapsulation of Lactobacillus plantarum in HIPEs. The freeze-dried powder

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of Lactobacillus plantarum was firstly suspended in grape seed oil at 1:40 w/v, to

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obtain a visually homogenous suspension before homogenization with the WPI

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microgels28. In the encapsulation study, the influence of different WPI concentrations

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with respect to the aqueous phase (0.5 wt%, 1.0 wt%, 4.0 wt% and 10.0 wt%),

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different pH values of the protein aqueous solution (pH 3.0, pH 4.0, pH 6.0 and pH

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7.0), as well as different volume fractions of grape seed oil (20%, 50%, 70%, and

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90%) were investigated systematically. The pH values of the WPI solutions were

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adjusted using hydrochloric acid or sodium hydroxide solutions. The oil and the WPI

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microgel solution mixtures were sheared at 10,000 rpm for 3 min (IKA T18 digital

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ULTRA TURRAX homogenizer, IKA Inc, Germany), to achieve the HIPEs. The

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control group was prepared by replacing WPI microgels with distilled water. The

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formed HIPEs were stored at 4 0C before measurements.

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Rheological Property of HIPEs. The rheological properties of the HIPEs were

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measured according to the methods in our previous study29. The rheological

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measurement of the resulting HIPEs was carried out with a rheometer (Anton Paar,

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Physica MCR 301, Austria) in oscillatory mode. A 25 mm-parallel steel plate

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geometry with a 500 μm gap was used. The strain sweep was performed at a fixed

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frequency of 1 Hz. The temperature was maintained at 25 0C.

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Characterization of the HIPEs Microstructure. The microstructure of HIPEs

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was studied by laser scanning confocal microscope (CLSM) according to the method

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reported before with minor modification30,31. The measurements were carried out

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using a SP8 confocal microscopy (Leica Microsystems Inc., Germany). HIPEs were

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colored by 5 mg/mL of dye solution (Nile Red in isopropyl alcohol), and the

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fluorescence emission of Nile Red was excited at 514 nm.

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Viability of Encapsulated Lactobacillus plantarum after Pasteurization.

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Lactobacillus plantarum-encapsulated samples were heated in a water bath, with

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temperature being monitored mimic the thermal pasteurization process to explore the

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survival ratio for probiotics after thermal treatment. The samples and control were all

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heated at 63 0C for 30 min in water bath32. Cell counts of the probiotics before and

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after pasteurization were enumerated using the method in Section Enumeration of

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Lactobacillus plantarum.

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Enumeration of Lactobacillus plantarum. Samples containing Lactobacillus

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plantarum were serially diluted to proper concentrations with sterile normal saline.

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Specifically, for samples without thermal treatment, the probiotics suspension was

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serially diluted to be 10-4, 10-5 and 10-6 times of the original concentration. While for

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samples subjected to the thermal treatment, the probiotics suspension was serially

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diluted to be 10-2, 10-3 and 10-4 times of the original concentration. After appropriate

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sample dilution, 1 mL of diluent was added to plates, followed by addition of MRS

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agar at about 45-50 0C. Plates were incubated at 30 0C for 48 h in thermostatic

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incubator before enumeration. The amounts of the colony forming unit in the range of

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30 to 300 were used for enumeration, which can be clearly accounted.

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Statistical Analysis. The ANOVA procedure was employed for statistical

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analyses, using software IBM SPSS Statistics v25.0. The differences between the

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trials were analyzed by Duncan test (p < 0.05).

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RESULTS AND DISCUSSION

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Effects of WPI Concentrations on Physicochemical Properties of the

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Microgels. Rather than adding chemical crosslinking agents, proteins can form

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microgels through denaturation, aggregation, electrostatic interaction, and formation

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of disulfide bonds during the thermal treatment, which is much more environmental

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friendly and beneficial for food safety concern. Schmitt et al reported that whey

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protein microgels were formed after heating 4.0 wt% protein solution at 85 0C in pH

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5.7-5.8 for 15 min33. However, the effect of protein concentration on the

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physicochemical properties of the microgels has not been clarified. In the present

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study, particle size, ζ-potential and morphology of the prepared WPI microgels were

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characterized with protein concentrations of 0.5 wt%, 1.0 wt%, 4.0 wt% and 10.0

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wt%.

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Fig. 1A showed the particle size, ζ-potential and polydispersity index (PDI) of

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the dispersion for WPI microgels in aqueous phase. With the increase of protein

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concentration, particle size of the WPI microgels decreased from 296.63 ± 3.07 nm to

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209.67 ± 1.56 nm, along with the decrease of PDI values. PDI value is a parameter

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indicating the size distribution of colloidal nanoparticles. Normally, PDI value less

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than 0.3 suggested a relatively homogeneously dispersed colloid suspension. A

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smaller PDI value refers to a more homogeneous dispersion. The phenomenon shown

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in the present study indicated that crowded and compact protein molecules in high

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concentration environments facilitated the formation of WPI microgels with smaller

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size and narrow size distribution. Besides, no significant change in ζ-potential could

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be observed with the increase of protein concentration. Fig. 1B showed that WPI

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microgels dispersed homogeneously as evidenced by DLS measurements. The

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morphologies of WPI microgels prepared with 0.5 wt%, 4.0 wt% and 10.0 wt%

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protein concentrations were shown in Fig. 1C-E. After thermal treatment, compact

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protein microgels with relatively uniform sizes were formed. Similar results were also

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reported in previous studies24. In consistent with the particle size results obtained

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from the DLS measurements, particle size of the microgels detected by TEM also

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decreased with the elevation of protein concentrations.

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Effects of WPI Solution pH on the Physicochemical Properties of the

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Microgels. Proteins adopt various structure conformations and have different

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electrostatic charges in different pH conditions. The effect of pH on the

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physicochemical properties of the formed WPI microgels deserves investigations. In

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the present study, the WPI microgels formed under pH of 3.0, 4.0, 6.0 and 7.0 were

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investigated for their physicochemical properties, including particle size, ζ-potential,

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size distribution and particle morphology, as shown in Fig. 2A. The particle sizes of

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the WPI microgels firstly increased from 309.40 ± 3.39 nm to 359.47 ± 3.00 nm as the

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pH value approached the isoelectric point of WPI (~pH 4.5). Further increase of pH

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value caused the particle size of the microgels drop to 278.23 ± 0.81 nm. Meanwhile,

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the ζ-potential of the WPI microgels went through 0, from + (30.9 ± 1.1) mV to –

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(36.4 ± 1.2) mV. According to a previous study at no-salt condition, stable

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suspensions of roughly spherical protein microgel particles with an average

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hydrodynamic radius of about 200 nm could be formed by heating -lactoglobulin in

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a narrow pH range of 4.6 to 5.8 34. In addition, it was reported that weak gels could

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form by heating the whey protein polymers at pH 6.0 to 8.035. Fig. 2B illustrated that,

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similar to Fig. 1B, the size distribution of WPI microgels under different pH

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conditions was also homogeneous based on DLS measurement. Fig. 2C-E indicated

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the morphology of the WPI microgels prepared at pH 4.0, pH 6.0 and pH 7.0, which

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were compact particles with nanoscale sizes. In consistence with the particle size

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results measured by DLS (Fig. 2A), the size of the protein microgels shown in Fig.

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2C-E decreased with the increase of the tested pH values.

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The Appearance of the HIPEs. The freeze-dried Lactobacillus plantarum

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powder was originally dispersed uniformly in grape seed oil, then further

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homogenized with the WPI microgel solution. Fig. 3A interpreted the effect of

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different WPI microgel concentrations (0.5 wt%, 1.0 wt%, 4.0 wt% and 10.0 wt%) on

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the appearance and properties of the emulsions with an internal oil phase volume

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fraction of 80%. It is obvious that the emulsion systems containing 0.5 wt% or 1.0

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wt% of WPI microgels could not stand the inversion experiment. While contrarily,

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addition of 4.0 wt% or 10.0 wt% of WPI microgels enabled the formation of stable

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HIPEs. The formulated HIPEs were homogenous gels, as revealed from their tilting

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stability. At the same protein concentration of 4.0 wt%, the HIPEs stabilized with

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WPI microgels prepared under pH 4.0, pH 6.0 and pH 7.0 environments could also

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stand the tilting test (Fig. 3B). However, the one prepared at pH 3.0 condition failed

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to pass the inversion experiment (Fig. 3B), indicating the strong acidity environment

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might impede the microgel formation34,35. The oil volume fraction also dramatically

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influenced the formation and physical properties of the emulsions. In the present

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study (Fig. 3C), the emulsion systems with oil volume fractions of 80% and 90%

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were homogenous gels, as presented from their tilting stability. However, the ones

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with oil volume fractions of 20%, 50% and 70% could not stand the inversion

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experiment, which were normal Pickering emulsions. The normal Pickering

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emulsions formed a creamed layer at the top of the vessel within minutes coexisting

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with a subnatant aqueous phase containing part of the protein microgels that did not

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take part in stabilizing the oil droplets (data not shown). Higher oil volume fraction

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resulted in more creamed layer and less subnatant aqueous phase. Similar

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phenomenon were also shown in previous studies27, 36.

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Rheological Properties of the Lactobacillus plantarum-Encapsulated HIPEs.

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The stability of the prepared emulsions against large amplitude oscillatory shear was

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tested by rheological measurements. Fig. 4 showed the strain dependence of the

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storage modulus (G') and the loss modulus (G") at a fixed frequency (1 Hz) for the

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emulsions formulated with different WPI microgel concentrations, pH values and

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grape seed oil volume fractions. According to the appearance of emulsions, the

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samples prepared with WPI microgels at concentrations higher than 1.0 wt% and with

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pH values higher than 4.0 were selected for the rheological measurements.

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Considering Fig. 4A and B, for all the tested samples, G' were larger than G" in a

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linear region at low strain rates, indicating the elastic (solid-like) behavior of sample

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gels. Nevertheless, G" became larger than G' when the strain rate was increased to

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sufficiently high levels (＞100%), revealing the viscous (liquid-like) behavior therein.

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The gel strength of the HIPEs enhanced with the elevation of WPI microgel

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concentrations (Fig 4A) as well as the pH values (Fig 4B), at which conditions the

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microgels were prepared. For the tested emulsion samples formulated with different

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oil volume fractions (Fig. 4C), only the one containing 90% of oil phase exhibited

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typical gel properties. In this case, G' were larger than G" in a linear region under low

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strain rates, and G" became larger than G' when the strain rate was elevated to

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sufficiently high level (＞100%). However, for the samples with oil volume fractions

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of 50% and 70%, G' and G" almost coincided with each other in a linear region under

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low strain rates, and G" became larger than G' when the strain was increased to

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sufficiently high level ( ＞ 100%). These results are in consistent with the gelling

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condition of the samples shown in Fig. 3C. Besides, encapsulation of the freeze-dried

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Lactobacillus plantarum powder didn’t change the viscoelasticity of the HIPEs

296

samples obviously (Supplementary Materials).

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Characterization of the HIPEs Microstructure by CLSM. The morphology of

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Lactobacillus plantarum-encapsulated HIPEs was investigated using CLSM by

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labeling WPI with Nile Red in water phase. The Nile Red-associated WPI microgels

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generate red color when excited by a laser with a wavelength of 514 nm. The

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microstructures of HIPEs containing 80% oil with different WPI microgel

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concentrations (1.0 wt%, 4.0 wt%, and 10.0 wt%) were shown in Fig. 5A-C. The oil

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droplets became denser and smaller with the elevation of WPI concentrations. Fig.

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5D-F showed the microstructures of the HIPEs stabilized with WPI microgels

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prepared at pH 4.0, pH 6.0 and pH 7.0. Fig. 5E and F suggested that the WPI

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microgels prepared at pH 6.0 and pH 7.0 formed a gel network in the continuous

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phase and confined oil droplets in the gel matrix. It is intriguing that the accumulation

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of WPI microgels on the surface of the oil droplets could be observed in Fig. 5D

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when the protein microgels were prepared at pH 4.0. This phenomenon would lead to

310

enhanced viscoelastic properties of the adsorbed polymer layer, which could further

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benefit the increase of HIPEs stability. Similar phenomenon was also observed in

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previous study, which might be related to the size, amphiphilicity and surface charge

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of the prepared microgels28. With the increase of the internal oil phase volume

314

fractions, more WPI microgels took part in stabilizing the oil-water interface, leading

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to denser and smaller oil droplets. As presented in Fig 5I, highly dense and

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concentrated oil droplets with homogeneous dispersion were detected, enabling the

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formation of HIPEs. The gel network in the continuous phase observed in the

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microstructure of the HIPEs could inhibit creaming and phase inversion, while the oil

319

droplets trapped in the gel matrix were heterogeneous in size distribution. A similar

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structure was also observed by Li et al. in HIPEs formed with PNIPAM-co-MAA

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microgel particles presented in the continuous phase37.

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Viability of Encapsulated Lactobacillus plantarum after Pasteurization. Due

323

to the sensitivity of probiotics towards water, the HIPEs were proposed to enhance the

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viability of the encapsulated Lactobacillus plantarum, which provided an isolated

325

lipid environment for accumulation of the microorganisms. Pasteurization is widely

326

applied in food processing to ensure food quality and food safety by heating at 63 0C

327

for 30 min. Therefore, the viability of the Lactobacillus plantarum after pasteurization

328

treatment was evaluated in the present study, which was shown in Fig. 6. Generally,

329

pasteurization treatment caused partial death of the probiotics with significant

330

decrease in cell viability. However, compared with the oil-water mixture system

331

without using WPI microgels as the emulsifiers, the HIPEs encapsulation system

332

distinctly enhanced the viability of the Lactobacillus plantarum. In addition, the

333

viability of the encapsulated Lactobacillus plantarum increased with the elevation of

334

the WPI microgel concentrations (Fig. 6A). Besides, the highest viability of the

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microorganism appeared in the HIPEs stabilized with WPI microgels prepared at pH

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4.0 (Fig. 6B). It was revealed in Fig. 5D that the WPI microgels formed at pH 4.0

337

were inclined to accumulate on the oil droplet surface, which was also in accordance

338

with the results obtained in previous study 38. Considering these two phenomena, it

339

could be predicted that the accumulated WPI microgels on the oil-water interface

340

were beneficial for enhancing the resistant capability of the encapsulated probiotics

341

against the harsh environments. Apart from enhanced microgel concentration, the

342

viability of encapsulated Lactobacillus plantarum also increased with the elevation of

343

internal oil volume fractions (Fig 6C). According to the literature reported previously,

344

encapsulating Lactobacillus plantarum in oil can increase their survival rate after

345

pasteurization, and the exclusion of water molecules around bacteria is one of the

16


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346

mechanisms to reduce the death of bacteria during heating39. Therefore, the

347

characteristic high volume fraction of the internal oil phase is advantageous to

348

enhance the viability of the encapsulated probiotics.

349

In summary, HIPEs can be employed as a promising vehicle for improving the

350

viability of probiotics during food thermal processing, due to high oil phase fraction

351

and the accumulation of WPI microgel layer on the oil-water interface. WPI

352

microgels with the particles sizes around 300 nm were prepared through thermal

353

treatment of WPI solution without addition of chemical crosslinking agents. The

354

particles size of the microgels decreased with the elevation of protein concentrations

355

as well as the leaving away of pH value from the isoelectric point of the protein. WPI

356

microgels prepared with protein concentrations higher than 4.0 wt% in pH 4.0, pH 6.0

357

and pH 7.0 environments could readily emulsify the oil (volume fraction higher than

358

80 w/w%) to form homogenous HIPEs with tilting stability. The HIPEs thus formed

359

had a cellular and tunable pore structure that could resist mechanical perturbation.

360

Encapsulation of Lactobacillus plantarum with the HIPEs was successful to increase

361

the cell viability after pasteurization processing. And the protective effect was

362

enhanced with the elevation of the oil volume ratios and the increase of WPI microgel

363

concentrations. Under different pH conditions, the strongest protective effect

364

appeared at pH 4.0, when the WPI microgels accumulated on the oil droplet surface.

365

HIPEs stabilized with the WPI microgels showed high potential for enhancing the

366

stability of probiotics during food thermal processing.

17



367

ASSOCIATED CONTENT

368

Supporting information

369

The Supporting Information is available free of charge on the ACS Publications

370

website. Figure S1 for the strain dependence of the storage (G') and loss (G") moduli

371

of the HIPEs stabilized by the WPI microgels before and after encapsulating with

372

Lactobacillus plantarum.

373 374

ACKNOWLEDGEMENTS

375

This work was supported by the National Natural Science Foundation of China (No.

376

31871843, 31501488), the Natural Science Foundation of Jiangsu Province--

377

Outstanding Youth Foundation (BK20160075), the Fundamental Research Funds for

378

the Central Universities, China (KJQN201648, KYLH201601, KYYZ201804,

379

KYDZ201903) .

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494

Figure 1. Characterization of the whey protein isolate (WPI) microgels prepared with

495

different protein concentrations at pH 6.0. (A) Change of particle size, zeta potential

496

and polydispersity index of the WPI microgels with the increase of the protein

497

concentrations; (B) The particle size distribution of the WPI microgels at different

498

protein concentrations; The morphology of the WPI microgels with different protein

499

concentrations of 0.5 wt% (C), 4.0 wt% (D) and 10.0 wt% (E) at pH 6.0, which were

500

characterized by transmission electron microscope (TEM). Different lowercase letters

501

shown in Figures 1A indicate the significant differences (p < 0.05) detected by

502

Duncan test.

503

Figure 2. Characterization of the whey protein isolate (WPI) microgels prepared at

504

different pH conditions with the same protein concentration of 4.0 wt%. (A) Change

505

of particle size, zeta potential and polydispersity index of the WPI microgels prepared

506

at different pH conditions; (B) The particle size distribution of the WPI microgels

507

prepared at different pH conditions; The morphology of the WPI microgels prepared

508

at different pH conditions of pH 4.0 (C), pH 6.0 (D) and pH 7.0 (E) with the protein

509

concentration of 4.0 wt%, which were characterized by transmission electron

510

microscope (TEM). Different lowercase letters shown in Figures 2A indicate the

511

significant differences (p < 0.05) detected by Duncan test.

512

Figure 3. Characterization of the high internal phase emulsions (HIPEs). Photographs

513

of the HIPE samples (A) stabilized by the whey protein isolate (WPI) microgel

514

particles with different protein concentrations of 0.5 wt% , 1.0 wt%, 4.0 wt% and 10.0

515

wt% prepared at pH 5.5 with the oil volume ratio of 80%; (B) stabilized by the WPI

25



516

microgel particles prepared at different pH conditions of pH 3.0, pH 4.0, pH 6.0 and

517

pH 7.0 with the protein concentration of 4.0 wt% and the oil volume ratio of 80%; (C)

518

prepared with different oil volume fractions of 20%, 50%, 70%, 80% and 90% with

519

the WPI microgel particles prepared at protein concentration of 4.0 wt% and pH 6.0.

520

Figure 4. Strain dependence of the storage (G') and loss (G") moduli of the high

521

internal phase emulsions (HIPEs) stabilized by the WPI microgels measured at a

522

frequency of 1 Hz (A) with different protein concentrations of 1.0 wt%, 4.0 wt% and

523

10.0 wt% prepared at pH 6.0 with the oil volume ratio of 80%; (B) with the WPI

524

microgel particles prepared at different pH conditions of pH 4.0, pH 6.0 and pH 7.0

525

with the protein concentration of 4.0 wt% and the oil volume ratio of 80%; (C) with

526

different oil volume fractions of 50%, 70% and 90% with the WPI microgel particles

527

prepared at protein concentration of 4.0 wt% and pH 6.0.

528

Figure 5. Laser confocal microscope (CLSM) photographs of the Lactobacillus

529

plantarum-encapsulated emulsions with different whey protein concentrations of (A)

530

1.0 wt%, (B) 4.0 wt% and (C)10.0 wt% (aqueous phase pH 6.0 and the oil volume

531

fraction of 80%); with different aqueous phase pH at 4.0 (D), 6.0 (E) and 7.0 (F) (WPI

532

microgel concentration of 4.0 wt% and the oil volume fraction of 80%); and different

533

oil phase volume fraction of 50% (G), 70% (H) and 90% (I) (WPI microgel

534

concentration of 4.0 wt% and pH 6.0). Here, the emulsion systems with oil volume

535

fractions of 50% and 70% were normal Pickering emulsions, rather than high internal

536

phase emulsions.

537

Figure 6. Viable cell count of the Lactobacillus plantarum encapsulated by the WPI

26


Page 26 of 34

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538

microgels stabilized emulsions (pH 6.0) with different (A) WPI concentration of 0.5

539

wt%, 1.0 wt%, 4.0 wt% and 10.0 wt% with 80% oil phase before and after

540

pasteurization; (B) different aqueous phase pH at 3.0, 4.0, 6.0 and 7.0 with 80% oil

541

phase before and after pasteurization; (C) volume fraction of oil phase of 20%, 50%,

542

70%, 90% before and after pasteurization. Different lowercase letters indicate the

543

significant differences (p < 0.05) detected by Duncan test.

27



A

C

B

D

E

544 545

Page 28 of 34

Figure 1

28


Page 29 of 34


A

546

B

547 548 549 550 551 552 553 554

C

D

E

555 556 557 558 559 560 561 562 563

Figure 2

564

29



565

A

B

566 567 568

C

569 570 571 572 573 574

Figure 3

30


Page 30 of 34

Page 31 of 34


A

B

C a

575 576

Figure 4

31



A

B

C

D

E

F

G

H

I

577 578

Figure 5

32


Page 32 of 34

Page 33 of 34


579

A

580

B

581 582 583 584

C

585 586 587 588 589 590 591

Figure 6 H

I

592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610

33



611 612 613

Table of Contents

614 615 616 617 618

Freeze Drying

Heating

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Enhancing the Viability of Lactobacillus plantarum as Probiotics

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