Fabrication of whey protein/pectin double layer microcapsules for improving survival of Lacticaseibacillus rhamnosus ZFM231

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International Journal of Biological Macromolecules 242 (2023) 125030

Available online 25 May 2023

Fabrication of whey protein/pectin double layer microcapsules for

improving survival of Lacticaseibacillus rhamnosus ZFM231

Liang Chen

, Wen-Wen Qian

, Shaobo Zhou

, Tao Zhou

, Qing Gu

School of Food Science and Biotechnology, Zhejiang Gongshang University, Xiasha, Hangzhou, Zhejiang 310018, PR China

School of Science, Faculty of Engineering and Science, University of Greenwich, Central Avenue, Chatham ME4 4TB, UK

ARTICLE INFO

Keywords:

Microcapsule

Lacticaseibacillus rhamnosus

Gastrointestinal conditions

ABSTRACT

To improve the viability of Lacticaseibacillus rhamnosus ZFM231 strain in the gastrointestinal tract and exhibit

better probiotic effect, an internal emulsication/gelation technique was employed to encapsulate this strain

using whey protein and pectin as wall materials to fabricate the double layer microcapsules. Four key factors

affecting the encapsulation process were optimized using single factor analysis and response surface method-

ology. Encapsulation efciency of L. rhamnosus ZFM231 reached 89.46 ±0.82 %, the microcapsules possessed a

particle size of 172 ±1.80

m and ζ-potential of −18.36 mV. The characters of the microcapsules were assessed

using optical microscope, SEM, FT-IR and XRD analysis. It was found that after exposure to simulated gastric

uid, the bacterial count (log (CFU g

−1

)) of the microcapsules only lost 1.96 units, the bacteria were released

readily in simulated intestinal uid, reaching 86.56 % after 90 min. After stored at 4 ◦C for 28 days and 25 ◦C for

14 days, bacterial count of the dry microcapsules decreased from 10.59 to 9.02 and 10.49 to 8.70 log (CFU g

−1

respectively. The double layered microcapsules could signicantly increase the storage and thermal abilities of

bacteria. Such L. rhamnosus ZFM231 microcapsules could nd applications as ingredient of the functional foods

and the dairy products.

1. Introduction

Lacticaseibacillus rhamnosus is a widely concerned probiotic in the

lactic acid bacterial family, as it has shown the capacity to inhibit Hel-

icobacter pylori infection [1], prevent intestinal damage, and improve

immunity [2]. In order to achieve these probiotic effects, L. rhamnosus

needs to be colonized in the intestine to a certain amount [3]. However,

L. rhamnosus is sensitive to external factors, e.g., low pH of gastric acid

and high content of bile salt [4,5], which leads to a relatively low sur-

vival rate in intestine environment, thereby limiting its wide applica-

tion. In addition, the probiotics are easily damaged during storage and

transportation process.

Microencapsulation of probiotics has been demonstrated to be an

effective strategy for the protection of bacteria from their surrounding

environmental conditions, thereby helping them colonize in the intes-

tine [6]. Several studies have shown the benets of microencapsulation

on long-term storage stability and the protection of probiotics under

gastrointestinal conditions [7–9]. Several methods have been applied

for the encapsulation of probiotics, such as extrusion method [10],

emulsication method [11], and gelation method [12]. Although

internal emulsication/gelation technique has been commonly used for

the encapsulation of bioactive compounds [13], the study using this

method for the encapsulation of probiotic bacteria has been rarely re-

ported. Proteins, starch and polysaccharides are often used as encap-

sulation wall materials [14–16]. These materials should have the

characteristics of non-toxicity, no side effects, good biocompatibility,

good lm-forming property, and no reaction with core materials. Whey

protein, a by-product of cheese production, is a popular encapsulating

material, mainly containing β-lactoglobulin serum albumin and immu-

noglobulin [17]. Due to the amphiphilic nature of whey protein [18], it

can form good emulsions and anti-gastric hydrogels [19]. However, the

wall material with a single constituent is difcult to provide adequate

coating for the core materials, leading to a failure in the achievement of

specic effects and functions, such as resistance to the external degra-

dation and the long-term storage [20]. It was reported that use of the

complex wall materials by combining whey protein with poly-

saccharides could avoid the disadvantages of using a single wall material

[9]. Pectin is a good choice for this purpose, furthermore, it possesses a

variety of biological activities [21].

In our previous study, a strain of L. rhamnosus ZFM231 with good

* Corresponding author:

E-mail addresses: taozhou@zjgsu.edu.cn (T. Zhou), guqing@zjgsu.edu.cn (Q. Gu).

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

https://doi.org/10.1016/j.ijbiomac.2023.125030

Received 20 December 2022; Received in revised form 10 May 2023; Accepted 20 May 2023

International Journal of Biological Macromolecules 242 (2023) 125030

exopolysaccharide (EPS)-producing capacity was isolated and identied

[22]. The EPS produced by this strain possess good probiotic effect, such

as hypolipidemic and antioxidant activities, gut microbiota-regulating

and colitis-alleviating effects [7,22,23]. Therefore, it is important to

develop a method to improve the viability of L. rhamnosus ZFM231

strain in the gastrointestinal tract and increase its stability, so as to

provide better probiotic effect. To this end, an internal emulsication/

gelation technique was employed for the encapsulation of L. rhamnosus

ZFM231 strain. Herein, the fabrication of double layer L. rhamnosus

ZFM231 microcapsules using whey protein as the inner wall material

and pectin as the protective wall material is reported. The tolerance of L.

rhamnosus ZFM231 microcapsules in the simulated gastric uid (SGF),

the release in the simulated intestinal uid (SIF), storage and thermal

stabilities were also investigated.

2. Materials and methods

2.1. Cultivation of L. rhamnosus ZFM231

L. rhamnosus ZFM231 was isolated from fresh milk, and has been

deposited in the China Center for Type Culture Collection (CCTCC)

under accession number NO. CCTCC M 2019883. L. rhamnosus ZFM231

was inoculated on MRS agar, and cultured at 37 ◦C for 48 h. A colony

with good growth was selected and inoculated into MRS broth for 24 h at

37 ◦C. After 3 generations of activation, the bacteria were collected by

centrifugation (4 ◦C, 8000 rpm, 10 min), the concentration of bacterial

solution was adjusted to 10

–10

CFU mL

−1

, and inoculated into MRS

broth with 2 % (v/v) inoculation amount at 37 ◦C for 24 h.

2.2. Microencapsulation of L. rhamnosus ZFM231

Microencapsulation of the bacteria was carried out referring to a

published method with slight modications [24]. In brief, whey protein

solution (4 %–12 %) was stirred (800 rpm) at 45 ◦C for 2 h, and heated to

80 ◦C and stirred for another 30 min, then allowed to stand at 4 ◦C

overnight after cooling on an ice water bath. The bacteria were collected

by the centrifugation (10,000 rpm, 10 min) of above bacterial suspen-

sion, and the concentration of the bacteria was adjusted to about

–10

CFU mL

−1

with normal saline, which was named as free bac-

teria solution. The free bacteria solution was mixed with whey protein

solution in a ratio of 1:15 (v/v), which was named as bacteria-whey

mixture. The mixture of water and soybean oil with a certain ratio

(1:1–1:5, v/v) was named as water-oil mixture. The bacteria-whey

mixture was mixed with water-oil mixture at a ratio of 3.5:1, followed

by the addition of CaCl

(nal concentration 0.01 %, w/v) and gluco-

lactone (nal concentration 0.4 %, w/v). The resulting solution was

heated at 40 ◦C for a certain time (1–5 h) at a certain stirring speed

(200–1000 rpm). The precipitate was obtained by centrifugation at

8000 rpm for 6 min.

2.3. Single factor analysis

The effects of whey protein concentration (4 %, 6 %, 8 %, 10 %, 12

%), stirring speed (200, 400, 600, 800, and 1000 rpm), emulsication

time (1 h, 2 h, 3 h, 4 h and 5 h) and water-oil ratio (1:1, 1:2, 1:3, 1:4, 1:5)

on the encapsulation efciency were studied using single factor tests.

2.4. Response surface methodology (RSM)

On the basis of the results of single factor tests, the inuences of the

four factors, including whey protein concentration (A), stirring speed

(B), emulsication time (C) and water-oil ratio (D) on the encapsulation

efciency were further investigated by RSM. Design expert 10 software

was used for the experimental design and analysis, the levels of each

factor were shown in Table 1. The design included 27 experimental

points, in which the central experiments were repeated three times.

2.5. Selection of protective agent of microcapsules

During freeze-drying, wet single-layer microcapsules might be

damaged, resulting in a reduced encapsulation efciency. This reduction

can be improved by adding a protective agent to form another layer of

coating on the surface of microcapsules, which can also avoid the rapid

hydrolysis of whey protein by gastric acid in the stomach, thereby

protecting the bacteria. In this study, trehalose, pectin and gelatin were

screened to select the optimal protective agent. The solution of wet

Table 1

Results of response surface experiments.

No. Whey

protein (A)

Stirring

speed

(B)

Emulsication

time

(C)

Water/oil

ratio

(D)

EE (%)

1 -1 0 0 -1 71.68 ±

1.30

2 0 -1 1 0 63.54 ±

0.93

3 0 0 1 1 64.50 ±

0.76

4 0 1 0 1 71.96 ±

0.89

5 1 0 0 1 66.35 ±

1.21

6 0 0 0 0 84.50 ±

1.11

7 0 0 -1 -1 63.86 ±

1.39

8 0 0 0 0 85.00 ±

1.34

9 0 -1 0 -1 64.50 ±

0.98

10 0 -1 0 1 61.95 ±

0.90

11 -1 0 -1 0 79.96 ±

2.01

12 0 0 -1 1 74.46 ±

1.20

13 -1 -1 0 0 67.38 ±

0.98

14 0 1 0 -1 65.23 ±

0.90

15 0 0 0 0 85.97 ±

1.45

16 1 0 1 0 77.51 ±

1.20

17 0 1 -1 0 65.05 ±

1.24

18 -1 0 0 1 72.00 ±

1.69

19 0 -1 -1 0 68.39 ±

1.29

20 1 0 0 -1 64.96 ±

1.43

21 1 -1 0 0 61.90 ±

1.14

22 -1 0 1 0 69.90 ±

1.50

23 1 0 -1 0 64.40 ±

1.59

24 0 1 1 0 67.82 ±

1.23

25 -1 1 0 0 67.64 ±

0.88

26 0 0 1 -1 68.76 ±

0.80

27 1 1 0 0 70.43 ±

1.00

Level

(−1)

6 600 2 1:3

Level

(0)

8 800 3 1:4

Level

(1)

10 1000 4 1:5

Unit % rpm h v:v

L. Chen et al.

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Fabrication of whey protein/pectin double layer microcapsules for improving survival of Lacticaseibacillus rhamnosus ZFM231

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