Journal Pre-proof

Lacticaseibacillus paracasei JY025 fortified milk powder: In vitro digestion

characteristics and accelerated storage stability

Yu Shen, Jiaxin Zhang, Ming Ma, Yueling Tian, Xu Wang, Xinyan Yang, Chaoxin
Man, Xiaoyan Pei, Qianyu Zhao, Yujun Jiang

PII: S0023-6438(24)00216-0
DOI: https://doi.org/10.1016/j.lwt.2024.115937
Reference: YFSTL 115937

To appear in: LWT - Food Science and Technology

Received Date: 5 October 2023

Revised Date: 28 February 2024
Accepted Date: 1 March 2024

Please cite this article as: Shen, Y., Zhang, J., Ma, M., Tian, Y., Wang, X., Yang, X., Man, C., Pei,
X., Zhao, Q., Jiang, Y., Lacticaseibacillus paracasei JY025 fortified milk powder: In vitro digestion
characteristics and accelerated storage stability, LWT - Food Science and Technology (2024), doi:
https://doi.org/10.1016/j.lwt.2024.115937.

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1 Lacticaseibacillus paracasei JY025 fortified milk powder: in
2 vitro digestion characteristics and accelerated storage
3 stability

4 Yu Shena, Jiaxin Zhanga, Ming Maa, Yueling Tiana, Xu Wangb, Xinyan Yanga, Chaoxin Mana,

5 Xiaoyan Peib, Qianyu Zhao a* Yujun Jianga**

a
6 Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast

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7 Agricultural University, Harbin 150030, China.

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b
8 National Center of Technology Innovation for Dairy, Huhehaote 010000, China.
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9 *Corresponding authors: Qianyu Zhao (E-mail addresses: 389832745@qq.com; Tel numbers: 86-
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451-55191820).
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10

11 **Corresponding authors: Yujun Jiang (E-mail addresses: yujun_jiang@163.com; Tel numbers:

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12 86-13936660851).
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1 ABSTRACT

2 To clarify the reciprocal influence between the fortified bacterial powder of Lacticaseibacillus

3 paracasei JY025 (LBP) with milk powder matrix during the gastrointestinal digestion and storage

4 period, the digestion characteristics and storage stability of Lacticaseibacillus paracasei JY025

5 fortified milk powder (LFMP) were explored in this study by the in vitro simulated digestion and

6 the accelerated storage. The results showed that the milk powder matrix significantly improved the

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7 survival of JY025 during the simulated digestion (the highest improvement of 27%) and storage

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8 period (the highest improvement of 37%), which suggests the milk powder matrix can be an
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9 effective strategy to enhance the viability of probiotics during the digestion and storage period.
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Moreover, the digestion characteristics and the physicochemical properties of the milk powder
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11 matrix were changed due to the fortified LBP during the simulated digestion and storage period.
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12 The fortified LBP decreased the particle size of milk powder digest and promoted the hydrolysis of
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13 milk protein during digestion due to the effect of both maltodextrin (wall material) and bacteria. The
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14 fortified LBP also increased the particle size of milk powder matrix and decreased the Maillard

15 reaction of milk powder matrix during the storage period, which is closely related to the storage

16 condition.

17

18 Keywords: Probiotic fortified milk powder; Lacticaseibacillus paracasei; Probiotic survival; In

19 vitro digestion simulation; Storage physicochemical property.

20 1. Introduction

21 Lacticaseibacillus paracasei, as one of the important lactobacilli species, widely exists in

22 traditional dairy products and the human gastrointestinal tract (Silvia et al., 2015). It has been well-

23 documented by the in vivo experiments that Lacticaseibacillus paracasei has a variety of functional

24 properties, such as antibacterial, improving diarrhea, enhancing immunity, and lowering cholesterol

25 (Jahreis et al., 2022). Therefore, Lacticaseibacillus paracasei has an important application prospect

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26 in dairy and functional foods. Nowadays, increasing amounts of milk powder are added with

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27 probiotics hoping to improve their nutritional quality. Probiotic activity is considered a prerequisite

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28
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for probiotics to carry out their beneficial function, which is affected by the digestive system and
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29 storage conditions (Liu et al., 2017).
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30 It is well established that the activity of probiotic cells is constantly challenged during digestion
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31 by stress factors such as gastric acid, bile acid, and digestive enzymes when the probiotic is exposed
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32 to the gastrointestinal environment (Chen, Cao, Ferguson, Shu, & Garg, 2012). The effective
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33 probiotic strains should be able to tolerate the gastrointestinal environment, maintain their activity,

34 and attach to intestinal epithelial cells that in turn produce the expected health benefits (Amund,

35 2016). It has been reported that the survival of probiotics is influenced by the food matrix during

36 gastrointestinal digestion (Dafe, Etemadi, Dilmaghani, & Mahdavinia, 2017). However, it is

37 unknown whether milk powder, as the food matrix, will improve the survival of probiotics during

38 digestion. Meanwhile, the influence of probiotics on the digestion of milk powder matrix needs to

39 be further explored.

40 Furthermore, the survival of the probiotic bacteria is also affected by multiple factors during

41 storage, such as oxygen, temperature, humidity, and food matrix (Dafe et al., 2017; Guerin et al.,
42 2017). It is suggested that functional foods that contain probiotics should have at least 106 CFU/g

43 or mL probiotic activity before consumption. This can ensure that enough live probiotics reach the

44 intestine, and in turn exert their health benefits (Castro, Tornadijo, Fresno, & Sandoval, 2015).

45 Probiotics, as active substances, are usually fortified to milk powder in the form of bacterial powder

46 through the dry mixing manufacturing process (Nagachinta & Akoh, 2013). However, there are

47 limited studies on the stability change under harsh conditions without packaging and the interaction

48 mechanism of probiotic milk powder during storage. When exposed to the external environment,

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49 the reaction rate within food is increased and the decay of food quality is sharply decreased, which

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50 -p
resulted in widespread attention to the quality of food (Nicoli & Calligaris, 2018).
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51 Therefore, in this study, Lacticaseibacillus paracasei JY025 fortified milk powders (LFMP)
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52 were chosen as the research object to investigate their digestion characteristics in vitro and

53 accelerated storage stability. The survival rates of JY025, lipolysis rates, and protein hydrolysis rates
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54 of LFMP were determined in different digestion periods to explore the mutual influence between
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55 JY025 and milk powder matrix during the in vitro simulated digestion. Moreover, during the storage
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56 period (storage temperature, 25 or 37°C; relative humidity, 75%) the changes in JY025 survival and

57 physicochemical characteristics of LFMP were measured every 7 days for 4 weeks, which further

58 explored the change mechanism of milk powder stability during storage. This study will provide an

59 important theoretical basis for clarifying the health benefits of probiotic milk powder in the human

60 body and ensuring its nutritional quality during storage.

61 2. Material and method

62 2.1. Preparation of LFMP

63 Lacticaseibacillus paracasei JY025 was originally isolated from traditional fermented dairy

64 products of the Tibet Autonomous Region, which was stored at the Key Laboratory of Dairy Science

65 (KLDS), Ministry of Education, China. The preserved Lacticaseibacillus paracasei JY025 was

66 activated twice in an MRS medium to obtain activated strains. The activated bacteria solution was

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67 inoculated into MRS broth medium with 2% inoculation amount, and performed in high-density

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68 mass culture at 37°C for 16 hours. During fermentation, the pH was controlled at 6.5 with 0.1 mol/L
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69 NaOH. Subsequently, the fermented bacteria liquid was centrifuged at 5000 × g, 4°C for 10 minutes.
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The protectant maltodextrin, which was purchased from Sigma Aldrich (dextrose equivalent 16-
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70

71 20%, Sigma-Aldrich, Inc., Shanghai, China), was added to the bacterial mud according to the quality
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72 ratio of 1:1, and made into bacterial suspensions. The prepared suspension was pre-frozen at -80°C
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73 for 12 hours and then dried at -55°C (cold trap) and 0.8 mbar (vacuum degree) by vacuum freeze-
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74 dying (Alpha 1-4 LSC plus, Marin Christ, Osterode, Germany) for 24 hours. The number of viable

75 bacteria in the prepared bacterial powder of Lacticaseibacillus paracasei JY025 (LBP) was 1011

76 CFU/g by viable cell counts.

77 The blank control milk powder (BCMP) was produced in the pilot factory according to the

78 formula technology of commercial milk powder. The primary and secondary homogenization

79 pressures were set as 110 and 35 bar, respectively. The inlet and outlet temperatures of drying air

80 were controlled at 180 and 90°C, respectively. The milk powder samples were then subjected to a

81 secondary drying process through the fluidized bed. And the powder outlet temperature was
82 controlled below 30°C. The BCMP was mixed with LBP in a certain ratio by the three-dimensional

83 solid mixer (Turbula T2F, WAB Machinery Company Ltd, Muttenz, Switzerland). And the final

84 concentration of viable bacteria was adjusted to 107, and 109 CFU/g in the LFMP, which were named

85 as LFMP-7 and LFMP-9, respectively. The main components of BCMP, LFMP-7, and LFMP-9 are

86 shown in Table 1. The analysis of each sample was performed in triplicate to reduce as much as

87 possible experimental errors.

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88 Determination of zeta potential

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2.2.

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89 The zeta potential of BCMP, LBP, LFMP-7, and LFMP-9 was measured by Malvern Nano
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90 (ZS90, Malvern Instruments Ltd., Malvern, UK). The assay samples were diluted 500-fold with
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Milli-Q water. Subsequently, the dilution samples were put and measured in the test cell. The
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91

92 measurement of zeta potential was repeated three times independently for each sample.
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Observation of morphology
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93 2.3.
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94 The scanning electron microscope (SEM) (SU8010, Hitachi Company Tokyo, Japan) was used

95 to observe the morphology of sample granules. About 10 mg of the powder samples were spread

96 evenly on the conductive double-sided carbon adhesive tape stuck on the sample holder, and the

97 excess powder was gently blown off. The powder samples were gold sputtered with the ion

98 sputtering instrument for 2 × 30 seconds and then observed with an accelerating voltage of 5 kV.

99 All representative images were selected from at least three independent experiments with similar

100 results.
101 2.4. Simulation of gastrointestinal digestion in vitro

102 The simulation of in vitro digestion was carried out according to the method reported by

103 Minekus et al (2014) and Brodkorb et al (2019), which was performed in triplicate for each sample.

104 Twenty-five g of BCMP, LBP, LFMP-7, and LFMP-9 were dissolved in 225 mL of deionized water

105 to prepare the milk solution. As shown in Table 2, 30 mL of the prepared milk solution was mixed

106 with 1.25 × gastric electrolyte stock solutions, CaCl2(H2O)2, enzymes, HCl (5 mol/L, adjust mixture

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107 pH to 3.0) and water (pH 3.0) to obtain 60 mL of final volume. The gastric final mixture was cultured

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108 in the constant temperature shaker at 37°C, 100 rpm, for 2 hours to simulate the gastric digestion

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109
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stage. Subsequently, 60 mL of the gastric digesta was mixed quickly with 1.25 × intestinal
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110 electrolyte stock solutions, CaCl2(H2O)2, enzymes, NaOH (5 mol/L, adjust mixture pH to 7.0) and
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111 water (pH 7.0) to obtain 120 mL of final volume. The intestinal final mixture was cultured in the
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112 constant temperature shaker at 37°C, 100 rpm, for 2 hours to simulate the intestinal digestion stage.
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113 The samples (one tube per time point) were harvested at regular time intervals. The measurement
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114 of JY025 survival was performed immediately after collection of samples. The remaining collected

115 samples were kept at -20°C for further characterization of gastrointestinal digests.

116 2.5. The survival of JY025 in the simulated gastrointestinal tract

117 To determine the survival rate of JY025 in the gastrointestinal tract, the number of viable

118 bacteria was measured in the digestive fluid at regular time intervals. The digestive fluid was

119 gradient diluted to a suitable concentration and inoculated on MRS medium. The MRS plates were

120 incubated at 37°C for 48 hours and then counted after incubation. The survival rate of JY025 was

121 calculated as follows:

 𝑁
122 𝑆𝑢𝑟𝑣𝑖𝑣𝑎𝑙 𝑟𝑎𝑡𝑒 (%) = × 100
𝑁0

123 Where N is the number of viable cells (log CFU/g) at a specific time point and N0 is the number

124 of the initial viable cell number (log CFU/g) at the beginning. The measurement of JY025 survival

125 was repeated three times independently for each digested sample.

126 2.6. Characterization of gastrointestinal digests

127 2.6.1. Determination of particle size and zeta potential

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128 The gastric and intestinal digestion products were diluted to a suitable concentration and

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129
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measured as described in Section 2.2. The measurement of particle size and zeta potential were
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130 repeated three times independently for each digested sample.
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131 2.6.2. Measurement of protein hydrolysis

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The degree of protein hydrolysis was measured by the O-phthalaldehyde (OPA) method (Shen
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132
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133 et al., 2023). OPA (80 mg) was dissolved in 2 mL of methanol (purity ＞ 99%). Then, the solution

134 was mixed with 5 mL of sodium dodecyl sulfate (SDS) solution (200 g/L), 50 mL of borax solution

135 (0.1 mol/L), and 200 μL of β-mercaptoethanol. The mixture was made up to 100 mL with distilled

136 water to obtain the OPA reagent. 10 μL of the sample was mixed with 200 μL of OPA reagent and

137 reacted for 15 minutes under dark conditions. The absorbance of the mixture was performed at 340

138 nm by the spectrophotometer (Evolution 201, Thermo Scientific, Inc., Waltham, USA). The

139 measurement of protein hydrolysis was repeated three times independently for each digested sample.
140 2.6.3. Determination of lipolysis

141 Due to fat digestion occurring primarily in the small intestine, lipolysis was measured by the

142 pH titrator (ZDJ5, Leizi Company Shanghai, China) during the intestine digestion stage (Liang, Qi,

143 Wang, Jin & McClements, 2017). The pH of the sample was adjusted to 7 by 0.015 mol/L NaOH.

144 The amount of free fatty acids (FFA) released in the intestine digestion stage was calculated as

145 follows:

f
𝑉NaOH × 𝑀NaOH

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146 𝑀𝐹𝐹𝐴 = × 1000
𝑉𝑚𝑖𝑙𝑘

147 Where MFFA is the molar concentration of FFAs released (µmol/mL), VNaOH is the volume of

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148
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NaOH solution consumed by the titration (mL), MNaOH is the molar concentration of NaOH solution
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149 (0.015 mol/L), Vmilk is the volume of milk digested in the intestine digestion stage (2.355 mL). The
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150 measurement of lipolysis was repeated three times independently for each digested sample.
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151 2.7. Storage of milk powder samples

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152 The reaction rate within food was increased when exposed to the external environment.

153 Therefore, 400 g of the prepared powder sample (BCMP, LBP, LFMP-7, and LFMP-9) was reserved

154 in a desiccator containing saturated NaCl solution (relative humidity, 75%). The desiccators were

155 placed into a constant temperature incubator at either 25°C or 37°C (oxygen content ＜ 21%) for 28

156 days respectively to further explore the accelerated storage stability of probiotic milk powder. Each

157 sample was performed in triplicate for each storage condition. Strains survival and Physicochemical

158 characteristics of the sample were determined every 7 days.

159 2.8. The survival of JY025 during storage

160 The JY025 survival of LFMP and LBP samples under different storage conditions was

161 determined by colony counting as mentioned in Section 2.5. The measurement of JY025 survival

162 was repeated three times independently for each storage sample.

163 2.9. Characterization of physicochemical properties of milk powder during storage

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164 2.9.1. water activity

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165 The water activity of the sample was measured by the LabMaster-aw neo water activity

166
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instrument (Novasina Company, Lachen, Switzerland). The sample (2 g) was positioned inside the
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167 instrument. The measurement of water activity was repeated three times independently for each
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168 storage sample.

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169 Moisture content

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2.9.2.
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170 The moisture content of the sample was determined by the microwave moisture analyzer (HX-

171 Q10, Shanghai Huxi Company, Shanghai, China). The sample (1 g) was spread on the sample stage,

172 and then the lid of the instrument was closed. The measurement time was approximately 70 seconds.

173 The measurement of moisture content was repeated three times independently for the storage of

174 each sample.

175 2.9.3. Particle size

176 The particle size of the sample was measured by the laser diffraction particle size analyzer

177 (Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK). The sample to be tested was weighted
178 to 13 ± 0.1 g, and placed inside the sample pool. The particle size analyzer was adjusted to dry mode.

179 The measurement of particle size was repeated three times independently for each storage sample.

180 2.9.4. Solubility

181 Referring to the method of Ho et al., 5 g of the sample was dissolved in 95 mL of distilled

182 water (Ho, Ton, Gaiani, Bhandari, & Bansal, 2021). The mixture was centrifuged by 3000 × g for

183 10 minutes. the precipitate was obtained and washed with distilled water three times. Then, the

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184 precipitate was placed into a weighing dish and dried to a constant weight. The solubility of the

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185 sample was calculated as follows: -p(𝑚2 − 𝑚1 × 100)
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186 𝑋 = 100 −
(1 − 𝐵) × 𝑚
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187 Where X is the solubility of the sample (g/100g); B is the moisture content of the sample

188 (g/100g); m is the mass of the sample (g); m1 is the mass of the weighing dish (g); m2 is the dried
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189 mass of the weighing dish and insoluble matter (g). The measurement of solubility was repeated
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190 three times independently for each storage sample.

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191 2.9.5. Color

192 The spectropolarimeter (ZE6000, Nippon Denshoku Company, Tokyo, Japan) was used to

193 determine the change of color during the storage period. The instrument was calibrated by standard

194 plate before sample analysis. About 2 g of sample was determined in the dark environment. The

195 chromaticity values of the samples were characterized by L*, a*, b*, and ΔE* respectively. The

196 measurement of color was repeated three times independently for each storage sample.
197 2.9.6. Maillard FAST index

198 The Fluorescence Advanced Soluble Tryptophan (FAST) method is based on simple

199 fluorescence measurement of milk supernatant, often applied to monitor Maillard reaction in dairy

200 products (Desic & Birlouez-Aragon, 2011). The sample (0.5 g) was dissolved in 3.5 mL of distilled

201 water and then mixed with 3.5 mL of sodium acetate buffer (0.1 mol/L, pH 4.6). The mixture was

202 centrifuged at 4000 × g for 10 minutes. The supernatant was filtered and diluted for determination.

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203 The fluorescence spectrophotometer (F-7100, Hitachi Company, Tokyo, Japan) was set with a slit

204 width of 5 nm and a scanning speed of 2400 nm/minute. When the fluorescence intensity of

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205
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advanced Maillard products (FAMP) was measured, the excitation wavelength and emission
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206 wavelength were set to 330 nm and 420 nm respectively. When the fluorescence intensity of
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207 tryptophan (FTrp) was measured, the excitation wavelength and emission wavelength were set to 290
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208 nm and 340 nm, respectively. The FAST index was calculated as follows:
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𝐹𝐴𝑀𝑃
209 𝐹𝐴𝑆𝑇 = × 100%
𝐹𝑇𝑟𝑝
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210 The measurement of the Maillard FAST index was repeated three times independently for each

211 storage sample.

212 2.9.7. Peroxide value

213 The sample (1 g) was dissolved in 2 mL Milli-Q, and mixed with 10 mL of isooctane:

214 isopropanol solution (3:1, volume ratio). The mixture was vortexed and centrifuged at 3400 × g for

215 5 minutes. Then, 1 mL of the supernatant was mixed successively with 2.8 mL of methanol: n-

216 butanol solution (2:1, volume ratio), 15 μL of ammonium thiocyanate (3.94 mol/L), and 15 μL of

217 ferrous ion solution. The resultant solution was left at room temperature for 20 minutes, and
218 measured at the wavelength of 510 nm by the spectrophotometer (Evolution 201, Thermo Scientific,

219 Inc., Waltham, USA). The concentration of hydroperoxide (μmol/kg) in the sample was calculated

220 by the standard curve cumene hydroperoxide. The measurement of peroxide value was repeated

221 three times independently for each storage sample.

222 2.10. Data Analysis

223 The data were presented as mean ± standard deviation (SD) for each technical repeat. All data

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224 were analyzed for homogeneity of variance and normal distribution using Levene’s and

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225 Kolmogorov–Smirnov (K-S) tests, respectively. Moreover, data from the storage tests were
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226 analyzed using a two-way repeated measures analysis of variance (ANOVA) followed by post hoc
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tests (Bonferroni test) to access differences at each time point between groups. The data sets were
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227

228 tested for normal distribution and sphericity before the ANOVA analysis. Sphericity was tested using
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229 Mauchly’s Test of Sphericity and a Greenhouse–Geisser correction was used if Mauchly’s Test of
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230 Sphericity showed a P value of less than 0.05. The analysis of variance was performed to assess the
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231 data using SPSS 23.0 (IBM SPSS Inc., Chicago, USA). The significance level for analyses was set

232 at α = 0.05 (P < 0.05). GraphPad Prism 9 (GraphPad Software, Inc., San Diego, USA) was used to

233 generate the graphs.

234 3. Results and discussion

235 3.1. Surface morphology and zeta potential

236 As shown in Figure 1, the shape and external morphology of the sample could be revealed by

237 SEM. It was obvious that the BCMP, which was prepared by spray drying, presented a regular ball-
238 like shape. The LBP, which was prepared by vacuum freeze-drying, exhibited an irregular block

239 morphology. Moreover, LBP’s particle size was significantly reduced at low-magnification images

240 (100 × magnification) after solid-state three-dimensional mixing treatment. It can be inferred that

241 the particle size of bacterial powder decreased due to high shear force during the dry mixing process

242 (Frosch, Wyrwich, Yan, Popp, & Frosch, 2019).

243 However, the irregular block morphology was not observed under low-magnification images

244 (100 × magnification) in the LFMP that was prepared by mixing BCMP and LBP. JY025 could be

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245 observed at the high-magnification images (20,000 × magnification) in the LBP, but which were not

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246 -p
observed in the LFMP. As is well known, there are frequent collisions between particles each other
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247 and vessel walls during the mixing procedure. Every collision results in the transfer of interfacial
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248 charges, which in turn leads to the interaction and fusion between the particles (Kuang, Oliveira, &

249 Crean, 2010; Muranov et al., 2011). Moreover, in addition to being an appropriate wall material for
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250 bioactive compounds, maltodextrin is also an amorphous material. Maltodextrin particles have a
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251 strong tendency to agglomeration, which is prone to reunite with other particles due to the collision
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252 between particles to form agglomerates (Montes et al., 2011). The higher frequency of collisions

253 between molecules increases the agglomeration effect between maltodextrin with spherical

254 molecules (Pashminehazar, Kharaghani, & Tsotsas, 2016). Therefore, we speculated that LBP was

255 agglomerated with milk powder particles due to electrostatic attraction and the maltodextrin

256 agglomeration properties during the solid-state three-dimensional mixing treatment. The decrease

257 in LBP particle size due to high shear force during the mixing process also raised the reunion

258 between LBP and BCMP.

259 To further validate the aforementioned speculation about charge transfer, the zeta potential of
260 the BCMP, LFMP, and LBP were performed. As shown in Table 3, the absolute value of zeta

261 potential was lower in the LBP compared with other samples, which suggests the bacterial powder

262 has a lower surface charge (Jafarieh et al., 2014). Meanwhile, the BCMP and LFMP had a higher

263 absolute value of zeta potential compared with the LBP. The absolute potential value of milk powder

264 decreased significantly (BCMP>LFMP-7>LFMP-9) with the increase of LBP addition. This result

265 further verified that there was an interaction between the bacteria powder and milk powder particles.

266 Maltodextrin, as the wall material of bacterial powder, is widely distributed on the surface and inside

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267 of LBP. Related studies have shown that maltodextrin will interact with the surface protein, resulting

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268 -p
in the shielding of the surface charge and reducing the absolute value of the sample zeta potential
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269 (Chen, Chen, Wu, & Yu, 2016; Lan, Xu, Ohm, Chen, & Rao, 2018).
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270 3.2. The survival of strain in the simulated gastrointestinal tract

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271 In this study, the samples were subjected to sequential gastric and intestinal digestion by
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272 simulating the gastrointestinal digestion model in vitro. The parameters of simulated gastrointestinal
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273 digestion such as electrolytes, pH, enzymes, bile, and digestion time were carefully optimized based

274 on physiological data and evidence. To simulate the pH in the entire passage through the gastric and

275 intestinal phase in static conditions, the pH of 3.0 and 7.0 respectively were used for gastric and

276 intestinal digestion (Minekus et al., 2014; Brodkorb et al., 2019). As shown in Figure 2, the JY025

277 survival rate of LFMP and LBP significantly decreased as the digestion progressed. It is well known

278 that the survival of probiotic bacteria in the gastrointestinal tract is greatly influenced by gastric acid

279 and bile salts (Guerin, Kriznik, Ramalanjaona, Roux & Girardet, 2016; Liu, et al., 2017; Zhang et

280 al., 2022). The low pH values induce cell death. Bile salts accumulate in the cytoplasm, which leads
281 to protein aggregation and intracellular imbalance, eventually resulting in cell death (Sidhu, Lyu,

282 Sharkie, Ajlouni, & Ranadheera, 2020).

283 In addition, the survival rate of JY025 in the LFMP was significantly higher than that in the

284 LBP. Related studies have previously reported that milk protein and fat act as buffers and protective

285 agents during gastrointestinal digestion, which protects bacterial cells from acid and bile conditions

286 (Liu, et al., 2017; Zhang et al., 2022). The hydrophobic groups of milk protein preferentially interact

287 with the bacterial cells, which further promotes the adhesion of bacterial cells to protein molecules

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288 causing the bacterial cells to embed in the milk powder matrix, thereby inhibiting the degradation

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289 -p
of gastric acid (Yuan, Hu, He, Hu, & Liu, 2023). Moreover, peptides released by proteolysis, which
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290 exhibit high bile salt binding capacity, may protect probiotic bacteria against the hazard of bile salts
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291 (Guerin et al., 2016). Thus, we speculated that the milk powder matrix could improve the survival

292 of probiotics in the gastrointestinal tract.

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293 In this study, 5.38 log CFU/g of reduction was detected in the LBP during simulated
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294 gastrointestinal digestion. The viable bacteria number of LFMP-7 and LFMP-9 were only decreased
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295 by 1.82 log CFU/g and 2.61 log CFU/g, respectively. Advanced microencapsulation techniques have

296 also been used to improve the survival of probiotics during digestion in the gastrointestinal tract,

297 such as electrospinning and electrospray. It has been reported that the viable bacteria number of

298 microencapsulated probiotics is reduced by 1.5-2.81 log CFU/g after digestion (Bosnea, Moschakis,

299 & Biliaderis, 2014; Yuan et al., 2023). However, these encapsulation technologies have some

300 limitations including complexity and high cost in the manufacturing process. The results showed

301 that the milk powder matrix has equivalent protective effects to microencapsulation for probiotics.

302 As a functional dairy product, probiotic-fortified milk powder has high marketability and consumer
303 acceptability. From this, it can be inferred that milk powder matrix can be an effective strategy to

304 enhance the viability of probiotics in the gastrointestinal tract. Meanwhile, whether the probiotics

305 fortification would produce any significant changes in the gastrointestinal digestion process of the

306 milk powder matrix, also requires further exploration.

307 3.3. Characterization of gastrointestinal digest in different stages

308 3.3.1. Zeta potential

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309 As shown in Figure 3, the absolute zeta potential of gastrointestinal digest presented decreasing

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310
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trends at first and increasing ones afterward as digestion progressed. The gastric juice contains free
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311 ions and an acidic pH value, which will shield the surface charge of the milk powder sample due to
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312 electrostatic effects and protein aggregation (Gasa-Falcon, Odriozola-Serrano, Oms-Oliu, & Martín-
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313 Belloso, 2019). Therefore, the absolute zeta potential of digest showed a significant decreasing trend
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314 as gastric digestion progressed. During intestinal digestion, the absolute zeta potential of digest
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315 showed a significant increasing trend, which is related to the hydrolysis of samples by the digestive

316 enzymes (Sun, Wang, Sun & Guo, 2019). The hydrolysis of the sample leads to an increase in the

317 number of surface charges (Gasa-Falcon et al., 2019). The increasing negative charges of the sample

318 can also be attributable to the existence of anionic ingredients (such as bile salts, phospholipids, and

319 free fatty acids) in the digest (Mun, Kim, McClements, Kim, & Choi, 2017; Yuan et al., 2019).

320 However, there was no significant difference in the change of zeta potential between each

321 digested sample during the same digestion time. Based on the results of the powder zeta potential,

322 it could be known that LBP only carried a low charge and agglomerated with the milk powder matrix.

323 Moreover, the pH and the anionic composition within the static simulation gastrointestinal digestion
324 system are the main factors affecting the surface charge of the digests (Brodkorb et al., 2019).

325 Therefore, it is logically speculated that the fortification of LBP will not affect the changes in the

326 surface charge of LFMP and BCMP samples during the digestion process.

327 3.3.2. Particle size

328 During the simulated digestion of the gastrointestinal tract, the particle size of the digest

329 displayed a trend of rising first and then decreasing (Figure 4). Similar results have been reported

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330 by Halabi et al., (2022) under in vitro digestion. From this, it is speculated that the emulsified layer

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331 of the sample surface suffered different degrees of structural damage in gastric digestion. The
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exposed κ -casein is preferentially dissolved by pepsin, which induces the rapid aggregation of
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332
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333 casein micelles (Ma et al., 2023). The acidic conditions of gastric digestion also promote the

334 aggregation of casein micelles (Borreani, Llorca, Larrea, & Hernando, 2016). Therefore, the particle
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335 size of the digest increased significantly in the gastric stage. However, the particle size of digest
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336 decreased significantly in the intestinal stage, which is mainly due to the dissociation and re-
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337 dispersion of casein micelles (Gallier, Acton, Garg & Singh, 2016).

338 In addition, the results showed that the particle size of LFMP was lower than the BCMP during

339 the digestion time of 30-150 minutes, which may be closely related to the fortification of LBP.

340 Maltodextrin, as the wall material of LBP, could interact with milk proteins leading to a reduction

341 in the hydrophobicity of the sample surface, which indicate the sample solubility is increased during

342 the simulated gastrointestinal digestion (Mehr & Koocheki, 2020; Wang, Chen, Zhang, Yu, & Wang,

343 2021). Therefore, we speculated that the solubility of LFMP samples was increased due to the LBP

344 fortification compared with BCMP during the simulated gastrointestinal digestion, which increases
345 the protein hydrolysis degree of LFMP and further decreases the particle size of LFMP digest in the

346 gastrointestinal tract. Moreover, the particle size difference between BCMP and LFMP was

347 significantly reduced during the digestion time of 180-240 minutes. The decrease in particle size

348 difference is due to the increase in the degree of sample hydrolysis (Jeong & Shin, 2018).

349 3.3.3. Protein hydrolysis

350 To verify the above speculation that maltodextrin interacted with milk protein and changed

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351 protein digestion, the protein hydrolysis of the LFMP and BCMP digest were determined. As shown

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352 in Figure 5, the degree of protein hydrolysis represented an increasing trend as the digestion
-p
proceeded. The protein hydrolysis degree of LFMP was significantly higher than that of BCMP
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353
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354 during the digestion time of 60-240 minutes (LFMP-9>LFMP-7>BCMP), which further suggests

355 the fortification of LBP can improve the degree of protein hydrolysis during gastrointestinal
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356 digestion (Liu, Bayjanov, Renckens, Nauta, & Siezen, 2010; Broadbent et al., 2011). On the one
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357 hand, the improvement of protein digestibility may be closely related to the above-mentioned
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358 interaction between maltodextrin and protein (Mehr & Koocheki, 2020; Wang et al., 2021). While

359 on the other hand, it may be closely related to the Lactobacilli strain. Related studies show that

360 Lactobacilli have a complex proteolytic system, consisting of extracellular proteases, transport

361 systems, and intracellular peptidases (Ji, Ma, Xu & Agyei, 2020). The simulated incubation

362 temperature of the gastrointestinal tract (37°C) is conducive to the secretion of extracellular

363 proteases by Lactobacilli, which also makes high enzymatic activity of extracellular proteases

364 (Islam et al., 2020). Therefore, we speculated that the activity of extracellular protease secreted by

365 Lactobacilli continues to increase as gastrointestinal digestion proceeds, which further changes the
366 conformation of casein and improves the hydrolysis of milk protein in the gastrointestinal tract

367 (Dalto, Audet, Girard, & Matte, 2018).

368 3.3.4. Fat hydrolysis

369 It is well known that fats are hydrolyzed to free fatty acids in the intestine due to the action of

370 pancreatic lipase and bile salts (Verkempinck et al., 2018). Therefore, the release of FFA was

371 determined during intestine digestion. As shown in Figure 6, the release of FFA increased as the

f
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372 digestion progressed, and reached a steady-state condition within 2 hours, which was consistent

r
373 with previous results (Liang et al., 2017; Luo, Liu, Liu, Shen & Ren, 2020). It has been reported
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that maltodextrins can stick to the surface of oil droplets to prevent lipase adsorption or increase the
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374
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375 digest viscosity, which further affects the digestion of lipids (Rahmani-Manglano, Tirado-Delgado,

376 García-Moreno, & Guadix, 2022). However, the difference in fat hydrolysis between LFMP with
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377 BCMP was not significant under the same digestive period. From this, we speculated that the
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378 maltodextrin content within the LFMP samples had not reached the critical concentration threshold
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379 that could change the intestinal lipolysis rate.

380 3.4. The survival of JY025 during storage

381 Apart from gastrointestinal transportation, probiotics also lose their activity during storage due

382 to the oxygen, moisture, and temperature content in the storage environment (Dafe et al., 2017;

383 Guerin et al., 2017). The hydrogen peroxide formed by the interaction of oxygen and moisture has

384 adverse effects on the proteins, lipids, and DNA of probiotic strains (Dianawati, Mishra & Shah,

385 2016). Moreover, moisture causes the crystallization of carbohydrates and increases molecular
386 mobility, disrupting the integrity of the bacterial cell membrane and ultimately leading to bacterial

387 death (Strasser, Neureiter, Geppl, Braun, & Danner, 2010). Storage temperature, which is closely

388 related to molecular motion and diffusion, is the main factor that affects the rate of physicochemical

389 reaction (including but not limited to the above reactions) (Masum, Chandrapala, Huppertz,

390 Adhikari, & Zisu. 2020). As shown in Figure 7, the survival rate of JY025 decreased significantly

391 with the extension of the storage period. The survival rate of JY025 in LBP, LFMP-7, and LFMP-9

392 was reduced to 33%, 38%, and 37% respectively at the storage period of 28 days at 25°C. Meanwhile,

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393 the survival rate of JY025 in LBP and LFMP was reduced to 0% at the storage period of 21 and 28

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394 -p
days at 37°C, respectively. These results suggested that higher storage temperature was not
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395 conducive to the survival of probiotics. The formation rate of lipid hydroperoxides in probiotic cell
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396 membranes is higher at higher storage temperatures, which further leads to rapid bacterial death

397 (Coulibaly, Amenan, Lognay, Fauconnier, & Thonart, 2009).

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398 Moreover, the survival rate of JY025 in LFMP was significantly higher than that in LBP during
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399 storage. The presence of proteins in the milk powder matrix has been shown to delay the
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400 crystallization of carbohydrates, thereby reducing the risk of crystallized carbohydrates to bacterial

401 strains (Shrestha, Howes, Adhikari, Wood, & Bhandari, 2007). Amorphous carbohydrates within the

402 food matrix play an important role in limiting bacterial membrane lipid oxidation, protein folding,

403 and chemical reactions by providing an effective environmental barrier and thus minimizing

404 excessive molecular movement (Poddar et al., 2022). Therefore, it shows that the milk powder

405 matrix can slow down the decrease of strain survival to some extent during storage.
406 3.5. Characterization of physicochemical properties of milk powder during storage

407 3.5.1. Water activity and moisture content

408 Water activity and moisture content are the important powder characteristics, which determine

409 the flowability and storage stability of powders (Premi & Sharma, 2017). As shown in Figure 8, the

410 water activity and moisture content of LFMP and BCMP increased significantly with the extension

411 of storage time. The milk powder gains or loses moisture according to the relative humidity of the

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412 surrounding environment (Wilbey, 2011). From this, it can be speculated that the increase in water

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413 activity and moisture content is due to the high relative humidity in the storage environment. The
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results also showed that the water activity and moisture content increased significantly with the
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414
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415 increase in storage temperature (25°C to 37°C). The relevant study has reported that the rate of water

416 migration in the powder matrix increases with the increase in storage temperature, which leads to
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417 an increase in water activity and moisture content (Yu, Schmidt, Bello-Perez & Schmidt, 2008).
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418 Combined with the results of JY025 survival, it was further verified that the water activity and
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419 moisture content of LFMP correlated negatively with JY025 survival (Jiménez, Flores-Andrade,

420 Pascual-Pineda & Beristain, 2015).

421 Moreover, the results showed that the water activity and moisture content of LFMP-9 samples

422 were significantly lower than that of BCMP samples under the storage condition of 25°C.

423 Maltodextrin, as the wall material for LBP, reduced hygroscopicity within LFMP samples, further

424 indicating that maltodextrin is effective in improving sample stability during storage (Tang et al.,

425 2013; Valenzuela & Aguilera, 2015). However, the moisture content within the LFMP samples was

426 significantly higher than that of BCMP as storage time increased under 37°C storage conditions,
427 which thus was speculated that the higher moisture content in the LFMP was closely related to the

428 fortification of Lacticaseibacillus paracasei JY025. For Lactobacilli, the outer surface is covered

429 with exopolysaccharides (EPS), which perform various functions for bacteria, such as protection

430 from harsh environmental conditions and desiccation (Rajoka et al., 2022). During storage at high

431 temperatures, Lacticaseibacillus paracasei will metabolize to produce more EPS to resist harsh

432 environmental conditions (Zhang et al., 2023). Furthermore, the postbiotic components (such as

433 EPS and peptidoglycan) are released as bacterial death under high-temperature and humidity storage

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434 conditions, increasing the polysaccharide content of LFMP (Sharafi, Divsalar, Rezaei, Liu, &

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435 -p
Moradi, 2023). It is well known that polysaccharides contain significant amounts of hydrophilic
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436 groups, which further leads to the increase of moisture content in the LFMP (Li et al., 2021).
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437 3.5.2. Particle size and solubility

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438 As illustrated in Figure 9, the average particle size of LFMP and BCMP was increased
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439 significantly with the extension of storage time and increase of storage temperature (25°C to 37°C),
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440 which is closely related to the crystallization degree of lactose during storage. Higher moisture

441 content accelerates the conversion of amorphous lactose to α-lactose monohydrate, which increases

442 the stickiness of the powdered milk sample and further leads to aggregation as well as caking of the

443 powdered milk particles (Saxena, Adhikari, Brkljaca, Huppertz, & Chandrapala, 2021). The particle

444 size of LFMP and BCMP reached their maximum at 37°C stored at 28 days, which is consistent

445 with the previous research. Related research has suggested that moisture content and storage

446 temperature have a coupling effect on lactose crystallization (Thomsen, Lauridsen, Skibsted, &

447 Risbo, 2005). Moreover, the results also showed that the average particle size of the LFMP was
448 significantly higher than that of BCMP under the storage condition of 37°C, which suggested the

449 fortification of LBP would promote the agglomeration of milk powder particles during higher

450 temperature storage. From the above analysis results of water activity and moisture content, it can

451 be known that the increasing polysaccharide content of LFMP leads to the increasing moisture

452 content and aggregation between particles in the LFMP samples with the extension of storage time

453 (Li et al., 2021). Moreover, the maltodextrin in LBP also absorbs a large amount of moisture as

454 storage time increases, thereby increasing the viscosity between particles, causing particle

f
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455 aggregation and the larger particle size of the sample (Siccama, Wientjens, Zhang, Boom, &

r
456 Schutyser, 2023). -p
re
457 In addition, it is well known that powder particle size can influence powder solubility. Powder
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458 solubility is the most important functional property of food powders (Sila et al., 2015). As shown in

459 Figure 10, the solubility of LFMP and BCMP decreased significantly with the extension of storage
na

460 time and increase of storage temperature (25°C to 37°C). The solubility of the LFMP was
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461 significantly lower than that of BCMP under the storage condition of 37°C, which is inversely
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462 correlated with the average particle size. This is consistent with findings from Kleekayai, et al.,

463 (2022). The related study has elucidated that the surface energy of particles was decreased with the

464 increase of particles, which leads to a decrease in sample solubility.

465 3.5.3. Color

466 The color of LFMP and BCMP were characterized and quantified by the color difference meter

467 at different storage stages. The results are presented in Figure 11, in which the L* represents the

468 lightness/darkness chromaticity value, a* represents the redness/greenness chromaticity value, b*

469 represents the yellowness/blueness chromaticity value, and ΔE represents the overall color

470 difference of the sample. The results suggested that the L* and ΔE show a decreasing trend, and a*

471 showed an increasing trend with the extension of storage time and increase of storage temperature

472 (25°C to 37°C). The color of LFMP and BCMP gradually changes from milky yellow to brownish

473 yellow, which is consistent with the results of Cesa et al., (2015).

474 Moreover, there were significant differences (p<0.05) in color characteristics between LFMP

475 and BCMP samples during the storage period. LFMP had higher L*, ΔE, and lower a* compared to

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476 BCMP, which suggests that the LFMP samples have a low degree of color change (Sun, Lin &

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477 -p
Zhang, 2021). Studies have shown that the color change of milk powder samples is closely related
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478 to Maillard reactions (Nagachinta & Akoh, 2013). Maltodextrin, as the wall material of LBP, is a
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479 dextrose equivalent is 16-20%, indicating that the reducing sugar in maltodextrin accounts for 16-

480 20% of the total mass (Zhao, & Tang, 2016). Reducing sugar, as the raw material of the Maillard
na

481 reaction, has a positive correlation trend with its concentration and degree of Maillard reaction (Li
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482 et al., 2020). This suggests that Maillard browning reaction activity between maltodextrin and milk
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483 protein is lower than that of lactose (McEwen, McKenna, O’Kane, Phillips, & Johns, 2010).

484 Moreover, the Maillard and lipid oxidation pathways are closely interrelated, which have common

485 intermediates and polymerization mechanisms (Zamora, & Hidalgo, 2005). It was therefore

486 speculated that the fortification of LBP would affect the degree of Maillard reaction and lipid

487 oxidation within milk powder samples, thereby affecting the color of samples.

488 3.5.4 Maillard reaction and lipid oxidation

489 The Maillard reaction often occurs in milk powder after long-term storage due to its rich protein
490 and reducing sugar, which will affect the nutritional quality of milk powder (Van Boekel, 1998).

491 Related studies have shown that the Maillard reaction is influenced by multiple factors such as

492 storage time, temperature, and water activity (Kirdponpattara, Phisalaphong, & Kongruang, 2017).

493 As shown in Table 4, the Maillard FAST index of LFMP and BCMP was increased significantly

494 with the extension of storage time and increase of storage temperature (25°C to 37°C). Moreover,

495 the Maillard FAST index of LFMP was lower compared to the BCMP with the extension of the

496 storage period, which further verified the above speculation that maltodextrin, as the wall material

f
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497 of LBP, will affect the Maillard reaction in the sample due to its low glucose equivalent (McEwen

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498 -p
et al., 2010). It is well known that lactose, as reducing sugar in dairy products, is the main substrate
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499 of the Maillard reaction and is also utilized in the metabolism of lactic acid bacteria (Rhimi,
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500 Chouayekh, Gouillouard, Maguin, & Bejar, 2011). Therefore, we speculated that lactose

501 competition was one of the main reasons for the decrease in Maillard reaction. In addition, from the
na

502 above results of JY025 survival, the bacterial activity was significantly reduced with the
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503 prolongation of storage time due to the influence of unfavorable storage factors (such as temperature,
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504 moisture, and oxygen). The bacterial cells are lysed after the strains death, which further results in

505 the release of various bacterial metabolic substances such as peptidoglycan (Mehta, Ayakar, &

506 Singhal, 2023). Relative studies have reported that peptidoglycan in the Lactobacilli cell wall binds

507 with Maillard by-products, thereby inhibiting the Maillard reaction (Kirdponpattara et al., 2017;

508 Shen et al., 2018). In summary, we speculated that the fortification of LBP could inhibit the Maillard

509 reaction in milk powder.

510 Peroxide value is an important index to evaluate lipid oxidation (Jiang et al., 2021). As shown

511 in Table 5, the peroxide value of LFMP and BCMP increased significantly with the extension of
512 storage time and increase of storage temperature (25°C to 37°C), which suggests that the high

513 temperature and water activity environment will promote the formation of lipid oxidation products

514 and increase the peroxide value of samples (Mahmoodani, Perera, Abernethy, Fedrizzi, & Chen,

515 2018). Moreover, it has been reported that lipid peroxides and fatty aldehydes produced by lipid

516 oxidation and degradation affect bacterial activity, which in turn leads to a decrease in the JY025

517 survival of LFMP samples with the extension of storage time and an increase in storage temperature

518 (Mafaldo et al., 2022). The production of lipid peroxides causes an increase in free radicals, which

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519 further damage the cell membranes of strains, thereby increasing cell permeability and reducing

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bacterial activity (Gerasymchuk, Robinson, Kovalchuk, & Kovalchuk, 2022). Fatty aldehydes
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521 inhibit the activity of Gram-positive bacteria by attacking functional proteins on the cell membrane
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522 (Patrignani et al., 2008). Therefore, we speculated that the degree of lipid oxidation was negatively

523 correlated with the survival of Lactobacilli.

na
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524 4. Conclusion
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525 This study took the LFMP as the research object to explore the in vitro digestion characteristics

526 and accelerated storage stability of probiotic fortification milk powder. The results showed that the

527 fortified LBP and milk powder matrix interacted and affected each other during the gastrointestinal

528 digestion and accelerated storage period. The milk powder matrix significantly improved the

529 survival of JY025 during the simulated digestion and storage period, which suggests the milk

530 powder matrix can be an effective strategy to enhance the viability of probiotics during the digestion

531 and storage period. Moreover, the digestion characteristics and the physicochemical properties of

532 the milk powder matrix were changed due to the fortified LBP during the simulated digestion and
533 storage period. The fortified LBP decreased the particle size of milk powder digesta and promoted

534 the hydrolysis of milk protein during gastrointestinal digestion due to the effect of both maltodextrin

535 (wall material) and bacteria, which will increase the subsequent absorption of milk protein.

536 Furthermore, the results suggested that the fortified LBP also affected the physicochemical

537 properties of the milk powder matrix during the storage period, which increased the particle size of

538 the milk powder matrix, and decreased the solubility and Maillard FAST index of milk powder

539 matrix during the storage period. This indicated that the fortified LBP had negative and positive

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540 effects on the storage stability of milk powder, which is closely related to the storage condition.

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This study helps clarify the reciprocal influence between probiotic strains and milk powder
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542 matrix during in vitro digestion and storage. Furthermore, it lays the foundation for finding effective
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543 methods to increase the utilization of digestive products and storage stability in probiotic-fortified

544 milk powder. It is necessary to mention that this study has faced its limitations. First of all, the static
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545 simulation cannot entirely replicate the dynamic complexity of human digestion. Secondly, based
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546 on the accelerated storage stability, the optimal storage conditions for probiotic-fortified milk
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547 powder need to be further optimized. Moreover, the internal (external) mass transfer and the stability

548 changes in the mechanism of probiotic-fortified milk powder under different packaging methods for

549 long-term storage are also what we would like to focus on in the future.

550 CRediT authorship contribution statement

551 Yu Shen: Investigation, Methodology, Data curation, Validation, Writing-original draft. Jiaxin

552 Zhang: Formal analysis, Validation, Writing-review & editing. Ming Ma: Data curation, Writing-

553 review & editing. Yueling Tian: Formal analysis, Writing-review & editing. Xu Wang:
554 Methodology, Data curation. Xinyan Yang: Methodology, Writing-review & editing. Chaoxin Man:

555 Data curation, Project administration. Xiaoyan Pei: Formal analysis, Validation. Qianyu Zhao:

556 Writing-review & editing, Supervision, Project administration. Yujun Jiang: Conceptualization,

557 Supervision, Project administration.

558 Acknowledgment

559 This work was supported financially by the National Center of Technology Innovation for

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560 Dairy under the project 2022-JBGS-1 entitled ‘Formation patterns of by-products of infant formula

r
561 milk powder processing and their effects on quality’.
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1 Table 1. The ingredients of BCMP, LFMP-7 and LFMP-9.

Component per kg BCMP LFMP-7 LFMP-9

Protein (g) 180 180 178

Fat (g) 132 132 131

Carbohydrate (g) 608 608 612

Sodium (mg) 3950 3950 3911

Calcium (mg) 13000 12999 12870

Iron (mg) 75 75 74

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Zinc (mg) 110 110 109

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Taurine (mg) 375 375 371

r
Vitamin A (μgRE) 4850 4850 4802

Vitamin D (μg)
-p
62 62 61
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Vitamin E (mg α-TE) 23 23 23
lP

Vitamin k (μg) 400 400 396

Thiamine (mg) 8 8 7
na

Vitamin B2 (mg) 7 7 6

Vitamin C (mg) 350 350 347

ur

2 BCMP: Blank control milk powder; LFMP-7: 107 viable Lacticaseibacillus paracasei JY025
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3 fortified milk powder; LFMP-9: 109 viable Lacticaseibacillus paracasei JY025 fortified milk

4 powder.
5 Table 2. The in vitro digestion experiment with 30 mL of milk solution.

Input 30 mL milk solution

Digestion phase Gastric (SGF) Intestinal (SIF)
Food or digesta 30 60
1.25 × electrolyte
stock solutions 24 48
(mL)
CaCl2(H2O)2
0.015 0.12
(0.3 mol/L) (mL)
Pancreatic
Enzymes Pepsin Trypsin Bile salts
lipase
Enzyme activity

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(U/mL) or bile
concentration 2000 U/mL 100 U/mL 2000 U/mL 10 mmol/L
(mmol/L) in total

r
digesta
pH adjustment
-p
HCl (5 mol/L) adjusted pH to 3.0 NaOH (5 mol/L) adjusted pH to 7.0
re
H2O (mL) Water with the corresponding pH was added until the final volume.
Final volume (mL) 60 120
lP

6 One unit of enzyme activity (U) is defined as the amount of enzyme that converted 1 μmol of

7 substrate to product in per min under standards conditions. U/mL refers to the enzyme activity per
na

8 milliliter of digesta.
ur
Jo
9 Table 3. The absolute zeta potential of LFMP, BCMP, and LBP.

Absolute zeta potential

Sample
(mV)

LFMP-7 23.3 ± 0.4C

LFMP-9 22.4 ± 0.4B

BCMP 24.4 ± 0.6D

LBP 2.4 ± 0.3A

10 Data are presented as means ± SD for samples with three replicates. Significant differences between

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11 different samples (P < 0.05) are indicated by capital letters. BCMP: Blank control milk powder;

LBP: Bacterial powder of Lacticaseibacillus paracasei JY025; LFMP-7: 107 viable

r
12

13
-p
Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9: 109 viable Lacticaseibacillus
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14 paracasei JY025 fortified milk powder.
lP

15
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ur
Jo
16 Table 4. The Maillard FAST index of LFMP and BCMP during the storage.
FAST index (%) 25℃ 37℃
Pooled SD
Storage days LFMP-7 LFMP-9 BCMP LFMP-7 LFMP-9 BCMP
1 7.5 7.5 7.4 7.5 7.5 7.4 0.5
7 7.7 7.6 8.1 8.5 8.2 9.1 0.2
14 8.4 8.2 8.6 9.4 8.8 8.9 0.4

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21 8.8 8.6 9.4 9.4 10.0 9.7 0.2
28 8.7 8.7 9.9 10.4 10.5 10.7 0.2

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Pooled SD 0.3 0.3 0.2 0.4 0.3 0.2 -

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Sample Effect F = 6.07; P = 0.01
Temperature Effect F = 12.04; P < 0.01

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Sample*Temperature Effect F = 8.50; P < 0.01

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17 Pooled SD: pooled standard deviation. BCMP: Blank control milk powder; LFMP-7: 107 viable Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9:

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109 viable Lacticaseibacillus paracasei JY025 fortified milk powder.
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19 Table 5. The peroxide value of LFMP and BCMP during storage.

20 Peroxide value 25℃ 37℃

Pooled SD
Storage days LFMP-7 LFMP-9 BCMP LFMP-7 LFMP-9 BCMP
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1 257 263 262 257 263 262 9
22
7 279 273 287 319 265 253 9
23 14 299 286 298 337 286 312 12

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24 21 319 283 302 341 330 323 12
28 314 332 327 327 318 323 12

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Pooled SD 9 10 8 9 9 14 -

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26 Sample Effect F = 1.41; P = 0.30

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27 Temperature Effect F = 2.99; P = 0.08
Sample*Temperature Effect F=2.14; P = 0.15

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28

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29 Pooled SD: pooled standard deviation. BCMP: Blank control milk powder; LFMP-7: 107 viable Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9:
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30 109 viable Lacticaseibacillus paracasei JY025 fortified milk powder.
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1
2 Figure 1. The morphology of BCMP, LBP, and LFMP correspond to a magnification of 100 ×

3 and 20000 ×. BCMP: Blank control milk powder; LBP: Bacterial powder of Lacticaseibacillus

4 paracasei JY025; LFMP-7: 107 viable Lacticaseibacillus paracasei JY025 fortified milk powder;

5 LFMP-9: 109 viable Lacticaseibacillus paracasei JY025 fortified milk powder.

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6
7 Figure 2. The JY025 survival rate of LFMP and LBP in the simulated gastrointestinal

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digestion. 4 h long in vitro digestion of samples with a gastric phase at pH 3.0 (0–120 min) and an
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9 intestinal phase at pH 7.0 (120–240 min). Significant differences between the same digestion times
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10 for the different samples (P < 0.05) are indicated by capital letters. LFMP-7: 107 viable
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11 Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9: 109 viable

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12 Lacticaseibacillus paracasei JY025 fortified milk powder; LBP: Bacterial powder of

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13 Lacticaseibacillus paracasei JY025.

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15 Figure 3. The zeta potential of LFMP and BCMP digesta during the simulated gastrointestinal

16 digestion. 4 h long in vitro digestion of samples with a gastric phase at pH 3.0 (0–120 min) and an

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intestinal phase at pH 7.0 (120–240 min). Significant differences between the same digestion times
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18 for the different samples (P < 0.05) are indicated by capital letters. LFMP-7: 107 viable
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19 Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9: 109 viable

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20 Lacticaseibacillus paracasei JY025 fortified milk powder; BCMP: Blank control milk
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21 powder.
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22

23 Figure 4. The particle size of LFMP and BCMP digesta during the simulated gastrointestinal

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digestion. 4 h long in vitro digestion of samples with a gastric phase at pH 3.0 (0–120 min) and an
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25 intestinal phase at pH 7.0 (120–240 min). Significant differences between the same digestion times
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26 for the different samples (P < 0.05) are indicated by capital letters. LFMP-7: 107 viable
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27 Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9: 109 viable

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28 Lacticaseibacillus paracasei JY025 fortified milk powder; BCMP: Blank control milk
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29 powder.
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30

31 Figure 5. The protein hydrolysis of LFMP and BCMP digesta during the simulated

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gastrointestinal digestion. 4 h long in vitro digestion of samples with a gastric phase at pH 3.0 (0–
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33 120 min) and an intestinal phase at pH 7.0 (120–240 min). Significant differences between the same
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34 digestion times for the different samples (P < 0.05) are indicated by capital letters. LFMP-
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35 7: 107 viable Lacticaseibacillus paracasei JY025 fortified milk powder; LFMP-9: 109
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36 viable Lacticaseibacillus paracasei JY025 fortified milk powder; BCMP: Blank control
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37 milk powder.
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38

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Figure 6. The fat hydrolysis of LFMP and BCMP digesta during the simulated gastrointestinal
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40 digestion. 2 h long in vitro digestion of samples with an intestinal phase at pH 7.0 (120–240 min).
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41 Significant differences between the same digestion times for the different samples (P < 0.05) are
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42 indicated by capital letters. LFMP-7: 107 viable Lacticaseibacillus paracasei JY025

43 fortified milk powder; LFMP-9: 109 viable Lacticaseibacillus paracasei JY025 fortified
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44 milk powder; BCMP: Blank control milk powder.

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45

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Figure 7. The JY025 survival rate of LFMP and LBP during the storage. Significant differences
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47 between the same storage times for the different samples (P < 0.05) are indicated by capital letters.
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48 LFMP-7 (25℃), LFMP-7 (37℃): 107 viable Lacticaseibacillus paracasei JY025

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49 fortified milk powder at 25℃ and 37℃, respectively; LFMP-9 (25℃), LFMP-9
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50 (37℃): 109 viable Lacticaseibacillus paracasei JY025 fortified milk powder at 25℃ and 37℃,
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51 respectively; LBP (25℃), LBP (37℃): Bacterial powder of Lacticaseibacillus

52 paracasei JY025 at 25℃ and 37℃, respectively.

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54

55 Figure 8. Water activity (a) and moisture content (b) of LFMP and BCMP during the storage.

56 Significant differences between the same storage times for the different samples (P < 0.05) are

57 indicated by capital letters. LFMP-7 (25℃), LFMP-7 (37℃): 107 viable

58 Lacticaseibacillus paracasei JY025 fortified milk powder at 25℃ and 37℃, respectively;

59 LFMP-9 (25℃), LFMP-9 (37℃): 109 viable Lacticaseibacillus paracasei JY025 fortified

60 milk powder at 25℃ and 37℃, respectively; BCMP (25℃), BCMP (37℃):

61 Blank control milk powder at 25℃ and 37℃, respectively.

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62

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Figure 9. The average particle size of LFMP and BCMP during the storage. Significant
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64 differences between the same storage times for the different samples (P < 0.05) are indicated
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65 by capital letters. LFMP-7 (25℃), LFMP-7 (37℃): 107 viable

66 Lacticaseibacillus paracasei JY025 fortified milk powder at 25℃ and 37℃, respectively;
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67 LFMP-9 (25℃), LFMP-9 (37℃): 109 viable Lacticaseibacillus paracasei

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68 JY025 fortified milk powder at 25℃ and 37℃, respectively; BCMP (25℃),
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69 BCMP (37℃): Blank control milk powder at 25℃ and 37℃, respectively.
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70

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Figure 10. The solubility of LFMP and BCMP during the storage. Significant differences
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72 between the same storage times for the different samples (P < 0.05) are indicated by capital letters.
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73 LFMP-7 (25℃), LFMP-7 (37℃): 107 viable Lacticaseibacillus paracasei JY025

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74 fortified milk powder at 25℃ and 37℃, respectively; LFMP-9 (25℃), LFMP-9

75 (37℃): 109 viable Lacticaseibacillus paracasei JY025 fortified milk powder at 25℃ and 37℃,
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76 respectively; BCMP (25℃), BCMP (37℃): Blank control milk powder at 25℃
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77 and 37℃, respectively.

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79
80 Figure 11. The L* (a), a* (b), and ΔE (c) of LFMP and BCMP during the storage. Significant
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81 differences between the same storage times for the different samples (P < 0.05) are indicated by
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82 capital letters. LFMP-7 (25℃), LFMP-7 (37℃): 107 viable Lacticaseibacillus

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83 paracasei JY025 fortified milk powder at 25℃ and 37℃, respectively; LFMP-9 (25℃),

84 LFMP-9 (37℃): 109 viable Lacticaseibacillus paracasei JY025 fortified milk powder at 25℃

85 and 37℃, respectively; BCMP (25℃), BCMP (37℃): Blank control milk powder

86 at 25℃ and 37℃, respectively.

1. JY025 and milk powder matrix interacted in the in vitro digestion simulation.

2. Milk powder matrix could improve the survivability of JY025 during digestion.

3. JY025 and milk powder matrix affect each other during the storage period.

4. Milk powder matrix could reduce the death of JY025 during the storage period.

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Dear editor:

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

Thank you and best regards.

Yours sincerely,

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Yujun Jiang

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