Journal Pre-proof

Probiotic dairy foods and postprandial glycemia: A mini-review

Laís C. Grom, Nathalia M. Coutinho, Jonas T. Guimarães, Celso F. Balthazar, Ramon

Silva, Ramon S. Rocha, Mônica Q. Freitas, Maria Carmela K.H. Duarte, Tatiana C.
Pimentel, Erick A. Esmerino, Márcia C. Silva, Adriano G. Cruz

PII: S0924-2244(20)30473-8
DOI: https://doi.org/10.1016/j.tifs.2020.05.012
Reference: TIFS 2857

To appear in: Trends in Food Science & Technology

Received Date: 22 December 2019

Revised Date: 9 May 2020
Accepted Date: 15 May 2020

Please cite this article as: Grom, Laí.C., Coutinho, N.M., Guimarães, J.T., Balthazar, C.F., Silva, R.,
Rocha, R.S., Freitas, Mô.Q., Duarte, M.C.K.H., Pimentel, T.C., Esmerino, E.A., Silva, Má.C., Cruz, A.G.,
Probiotic dairy foods and postprandial glycemia: A mini-review, Trends in Food Science & Technology
(2020), doi: https://doi.org/10.1016/j.tifs.2020.05.012.

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 Probiotic Dairy Foods and Postprandial glycemia : a mini-review

Laís C. Grom1, Nathalia M. Coutinho2, Jonas T. Guimarães2, Celso F. Balthazar2,

Ramon Silva1,2, Ramon S. Rocha1,2, Mônica Q. Freitas2, Maria Carmela K.H. Duarte2,

Tatiana C. Pimentel3, Erick A. Esmerino2, Márcia C. Silva1, Adriano G. Cruz1*

1
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ),

Departamento de Alimentos, 20270-021, Rio de Janeiro, Brasil

2
Universidade Federal Fluminense (UFF), Faculdade de Medicina Veterinária,

24230-340 Niterói, Brazil

3
Instituto Federal do Paraná (IFPR), Paranavaí, 87703-536, Paraná, Brasil

Running title: Post Prand. Prob. Dairy Foods

* email: food@globo.com/adriano.cruz@ifrj.edu.br (A.G. Cruz)

 1 Abstract

2 Background: Postprandial glycemia after food intake is considered the most

3 important parameter to control the risk of diabetes. Although probiotic dairy

4 products may have a regulatory effect on postprandial blood glucose, this topic

5 has been poorly addressed in scientific studies.

6 Scope and approach: This review aims to discuss the relationship between the

7 consumption of probiotic dairy products and the maintenance of postprandial

8 glycemia, presenting the mechanisms of action of probiotics and the impact of

9 the dairy matrix, the probiotic strain, and addition of ingredients, among others.

10 A comparison between the effects of dairy products and the synthetic drugs

11 conventionally used was also carried out. Finally, the review deals with

12 strategies for increasing the functionality of probiotic dairy products.

13 Key findings and conclusion: Ripened cheeses, fermented dairy products, and

14 whey-based products (milk beverages) containing probiotic strains with higher

15 proteolytic and EPS-forming capacities, and the addition of prebiotics and/or

16 plant-derived products have a greater effect on postprandial glycemic

17 regulation and/or inhibition of α-glucosidase and α-amylase enzymes. The

18 literature has focused on the in vitro inhibitory effect on digestive enzymes,

19 suggesting that clinical studies should be a priority. The inhibitory effect

20 observed for the probiotic dairy products can be quantitatively comparable to

21 that provided by synthetic drugs, depending on the dairy matrix and the

22 probiotic strain, with promising results.

23 Keywords: probiotics; dairy foods; postprandial glycemia.

 1 Probiotic Dairy Foods and Postprandial glycemia : a mini-review

3 Laís C. Grom1, Nathalia M. Coutinho3 , Jonas T. Guimarães2,, Celso F. Balthazar2,

4 Ramon Silva1,2, Ramon S. Rocha1,2, Mônica Q. Freitas2, Maria Carmela K.H. Duarte3,

5 Tatiana C. Pimentel3, Erick A. Esmerino2, Márcia C. Silva1, Adriano G. Cruz1*

1
7 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ),

8 Departamento de Alimentos, 20270-021, Rio de Janeiro, Brasil

2
9 Universidade Federal Fluminense (UFF), Faculdade de Medicina Veterinária,

10 24230-340 Niterói, Brazil

3
11 Instituto Federal do Paraná (IFPR), Paranavaí, 87703-536, Paraná, Brasil
4
12 Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ),

13 Curso de Farmácia, 21715-000, Rio de Janeiro, Brasil

14

15

16

17

18

19 Running title: Post Prand. Prob. Dairy Foods

20 * email: adriano.cruz@ifrj.edu.br (A.G. Cruz)

1
21 1. Introduction

22 Diabetes is a metabolic disorder characterized by insulin resistance or a

23 relative deficiency of insulin secretion, leading to an increase in blood glucose

24 levels (Chen et al., 2014a). There are several types of diabetes, including Type

25 1 diabetes (DM1, T-cell-mediated autoimmune disease, leading to the

26 destruction of insulin-producing pancreatic beta cells), Type 2 diabetes (DM2,

27 triggered by a genetic tendency associated with a routine without healthy

28 habits, resulting in insufficient insulin production and/or changes in its effects),

29 gestational diabetes (occurring during pregnancy and caused by dysfunction in

30 the production and action of insulin in the body), and other rarer types, such as

31 drug-induced endocrine diseases (Solis-Herrera et al., 2018). In 2013, 382

32 million people had diabetes worldwide, and this number is expected to rise to

33 592 million by 2035 and 629 million by 2045 (Kim, Keogh & Clifton, 2018, Kerry

34 et al., 2018). Several strategies have been used to reduce the risk and/or to

35 treat diabetes, such as the regular use of antidiabetic medications, diet

36 manipulation, changes in lifestyle, and regular exercise, aimed to keep blood

37 glucose (fasting and postprandial) at adequate levels (Patil et al., 2015).

38 The postprandial glucose management aims to measure the blood

39 glucose levels after food intake, and is considered the most important

40 parameter to control the risk of diabetes, which can lead to serious long-term

41 complications including hypertension, cardiovascular disorders, blindness, and

42 kidney impairment (Du & Myracle, 2018, Grom et al., 2020). One of the

43 strategies to keep postprandial glucose at adequate levels is the inhibition of

44 the α-amylase and α-glucosidase enzymes, which catalyze the digestive process

2
45 of carbohydrates. This inhibition leads to a reduction in glucose absorption and

46 consequently the postprandial glucose levels. Although administration of

47 synthetic α-glucosidase inhibitors is the main therapeutic approach, these drugs

48 are expensive and have side effects such as bloating, abdominal pain, vomiting,

49 and diarrhea (Chen et al., 2014a, Graham et al., 2019).

50 Therefore, there is a growing demand of health-conscious consumers

51 looking for foods that can help maintain blood glucose at appropriate levels

52 (Nikbakht et al., 2018). Probiotic dairy products may have a regulatory effect on

53 postprandial blood glucose (Grom et al., 2020); however, this topic has been

54 poorly addressed in scientific studies. To the best of our knowledge, this is the

55 first review that addresses the relationship between the consumption of

56 probiotic dairy products and the maintenance of postprandial glucose. This

57 review aims to discuss the relationship between the consumption of probiotic

58 dairy products and the maintenance of postprandial glycemia and/or inhibition

59 of α-amylase and α-glucosidase enzymes, presenting the mechanisms of action

60 of probiotics and the impact of the dairy matrix, the probiotic strain, and the

61 addition of ingredients, among others. In addition, it compares the effects

62 between dairy products and the conventionally used synthetic drugs. Finally, it

63 addresses strategies for increasing the functionality of probiotic dairy products.

64

65 2. Postprandial glycemia

66 Postprandial glycemia is the blood glucose levels after food consumption.

67 In healthy subjects, the glucose concentrations begin to rise about 10 minutes

68 after starting a meal, with a peak reached after 60 minutes (usually <140

3
69 mg/dL), then returning to preprandial levels within 2 to 3 hours (Gross, Ferreira

70 & Oliveira, 2003). Diabetes patients, however, have levels greater than 200

71 mg/dL after 60 minutes.

72 α-amylase (1,4-α-D-glucan-glucanohydrolase) is an enzyme that

73 hydrolyses polysaccharides to glucose and maltose oligosaccharides. α-

74 glucosidase is a membrane bound enzyme that is located in the epithelium of

75 the brush borders in the small intestines. This enzyme hydrolyses the

76 oligosaccharides at the non-reducing links and release the bound α-glucose,

77 thus increasing the glucose level in the serum (Sekar et al., 2019). The

78 inhibition of these enzymes delays and prolongs the digestion of carbohydrates,

79 thereby reducing the rate of glucose absorption and, consequently, the sudden

80 postprandial increase in plasma glucose (Balisteiro et al., 2013). Therefore,

81 several studies on postprandial glucose have measured blood glucose in vivo

82 after food intake or the inhibition of α-glucosidase and α-amylase activity in

83 vitro (Grom et al., 2020).

84

85 3. Probiotic Dairy Products and Health Benefits

86 Probiotics are living microorganisms which when administered in

87 adequate amounts confer health benefits to the host (FAO/WHO, 2001; Hill et

88 al., 2014). Probiotics should be kept viable throughout the product storage, and

89 may survive the adverse stomach conditions and colonize the gut, even

90 temporarily, by adhering to the human intestinal epithelium (Pereira et al.,

91 2018). Many studies consider a minimum initial dose of probiotics of 106-108

92 CFU/g to resist the gastrointestinal tract and reach the intestines in adequate

4
 93 quantities (Kim, Keogh & Clifton, 2018). However, the minimum amount

94 required and the optimal administration period of probiotics has not been fully

95 established, as the effect varies according to the microbial species and the type

96 of food (Gomand et al., 2019). Although the consumption of probiotic products

97 is safe, once studies have shown that the administration of probiotics to healthy

98 individuals does not increase the risk of disease, safety tests should be

99 performed for new probiotic strains (Pereira et al., 2018).

100 Dairy products are preferred food matrices for the addition of probiotics,

101 and the most studied strains are Lactobacillus acidophilus, Lactobacillus casei

102 and Bifidobacterium (Champagne, Daga & Cruz, 2018). Fermented dairy

103 products are the major vehicle for probiotics release as they have a positive

104 image to consumers, do not require changes in technology and manufacturing

105 process, and can protect probiotics through gastrointestinal transit. In addition,

106 the fermentation process acts to maintain the microbial viability, and consumers

107 are familiar with the presence of live microorganisms in these products (Boza-

108 Méndez, López-Calvo & Cortés-Muñoz, 2012; Gomand et al., 2019).

109 The health benefits associated with the consumption of probiotic cultures

110 include the control of intestinal infections, stimulation of gut motility with

111 consequent relief of constipation, improved absorption of certain nutrients,

112 improved use of lactose, relief of symptoms of lactose intolerance, decreased

113 cholesterol levels, anticarcinogenic effects, and stimulation of the immune

114 system. These benefits are specific to each strain, and a strain alone cannot

115 confer all the benefits (Kerry et al., 2018; Quigley, 2019; Cheng et al., 2019).

116

5
117 4. Probiotic Dairy Products and Postprandial Glycemia

118 The probiotic dairy products may help to reduce postprandial blood

119 glucose, and several mechanisms have been proposed, including the inhibition

120 of α-amylase and α-glucosidase enzymes (Chen et al., 2014a, Ayyash et al.,

121 2018; Figure 1), which can reduce the polysaccharide and disaccharide

122 hydrolysis and glucose absorption, thereby maintaining blood glucose levels

123 (Zeng et al., 2016). The inhibitory activity of probiotic dairy products on

124 digestive enzymes is due to the direct action of probiotic cultures or an indirect

125 action by the presence of bioactive peptides in the products. Probiotics act on

126 casein and whey proteins during the fermentation process, manufacture,

127 ripening and storage of dairy products, resulting in a higher concentration of

128 these compounds (Thakkar et al., 2018; Mushtaq et al., 2019). These bioactive

129 peptides are capable of binding to the enzyme active sites, decreasing the

130 enzyme activity (Wihansah et al., 2018). Other mechanisms include the use of

131 glucose by probiotic cultures, alteration of environmental conditions in the gut,

132 and stimulation of incretin secretion, which is a hormone released from

133 pancreas and gut, responsible for regulating glucose metabolism, resulting in

134 maintenance of postprandial glucose (Panwar et al., 2014, Chen et al., 2014b).

135 Studies about the relationship between the consumption of probiotic

136 dairy products and postprandial glycemia are scarce and have focused on in

137 vitro inhibition of α-glucosidase and α-amylase (Table 1). Only one study

138 effectively evaluated the postprandial glucose after dairy food consumption by

139 individuals (Grom et al., 2020). The effect of probiotic-enriched dairy

140 consumption on postprandial blood glucose is dependent on several factors,

6
141 including the type of product administered (Ayyash et al., 2018, Grom et al.,

142 2020, Apostolids et al., 2007), strain of probiotic used (Ayyash et al., 2018),

143 ingredients incorporated into the product (Shori & Baba, 2013, Su et al., 2018),

144 presence of prebiotic components in the formulation (Balthazar et al., 2019),

145 among others.

146 The inhibitory effect of the probiotic cultures on the postprandial

147 glycemia and/or inhibition of α-glucosidase and α-amylase enzymes is

148 dependent on the type of dairy food. The dairy foods commonly studied were

149 cheeses (Al-Dhaheri et al., 2017; Mushtaq et al., 2019; Wang et al., 2019;

150 Grom et al., 2020), fermented milk (Apostolidis et al., 2007; Ayyash et al.,

151 2018; Kinariwala et al., 2019; Graham et al., 2019), yogurts (Shori & Baba,

152 2013; Muganga et al., 2015; Amirdivani, 2015; Wihansah et al., 2018), and

153 Kefir (Du & Myracle, 2018).

154 Grom et al. (2020) evaluated the effect of consumption of different

155 probiotic dairy matrices (L. casei, 107-108 CFU/g, 50 g of Minas Frescal cheese,

156 50 g of Prato cheese, or 300 mL of dairy beverage associated with 50 g of

157 bread) on postprandial blood glucose in healthy subjects (n = 15, aged 22-46

158 years, and body mass index 18-24 kg/m2). The consumption of probiotic Prato

159 cheese + bread resulted in a lower increase in postprandial blood glucose (13

160 mg/dL) when compared to bread (19 mg/dL), probiotic Minas Frescal cheese +

161 bread (20 mg/dL) and probiotic dairy beverages + bread (30 mg/dL), indicating

162 that it may contribute to the reduction of postprandial glucose. The results were

163 associated with the good physicochemical characteristics (higher protein and

164 lipid content) of the matrix and the presence of a greater number of bioactive

7
165 compounds, resulting from the biochemical reactions during ripening (Grom et

166 al., 2020). Prato cheese is ripened for at least 25 days, and, during this

167 processing step, caseins are hydrolyzed by the residual coagulant, enzymes

168 naturally present in milk, and enzymes from starter and/or probiotic cultures,

169 leading to the release of peptides, including bioactive peptides which could

170 have inhibitory activity against α-glucosidase and α-amylase enzymes,

171 consequently, reducing the postprandial glycemia (Baptista et al., 2018). The

172 addition of probiotic cultures with proteolytic capacity can increase the release

173 of these bioactive compounds (Mushtaq et al., 2019). The consumption of dairy

174 beverages also resulted in a significant effect on postprandial blood glucose,

175 with a return to basal levels, probably due to the presence of whey proteins

176 and the probiotic effect on these proteins, with formation of bioactive peptides

177 (Grom et al., 2020).

178 The fermentation process has a significant effect on the product

179 functionality. Apostolids et al. (2007) reported an increase in α-glucosidase

180 inhibitory activity of fermented milks containing L. acidophilus (4% increase,>

181 106 CFU/g) during the fermentation process. Kinariwala et al. (2019) studied

182 the inhibitory activity of lactobacillus strains (L. fermentum M2 and M7) on the

183 digestive enzymes in fermented milks, and also observed an increased inhibition

184 of α-amylase activity during the fermentation process (24 h), with an increase

185 of 59.47% and 53.45% for the M2 and M7 strains, respectively. For α-

186 glucosidase activity, percentage increases of 7.09 and 8.36% were observed,

187 respectively. Therefore, the manufacture of fermented products (fermented

8
188 milks, yogurts, fermented dairy beverages, etc.) using suitable probiotic strains

189 can improve the functional properties of the products.

190 The effect of the type of milk on inhibition of digestive enzymes has been

191 contradictory in different studies. Ayyash et al. (2018) evaluated fermented

192 milks (cow or camel) containing different probiotic strains (Lb. reuteri

193 KX881777, Lb. plantarum KX881772, Lb. plantarum KX881779, and Lb.

194 plantarum DSM2468, 108 CFU/mL) and reported a higher inhibitory activity of

195 α-glucosidase (≈30-55%) and α-amylase (≈40-50%) of the fermented product

196 from bovine milk when compared to camel milk (≈ 30-40 and ≈ 25-40%,

197 respectively). In contrast, Shori & Baba (2011) reported a more pronounced

198 inhibition of α-amylase in probiotic yogurt (Lactobacillus acidophilus LA-5,

199 Bifidobacterium bifidum Bb-12, Lactobacillus casei LC-01, Lactobacillus

200 rhamnosus, Bifidobacterium infantis, and Bifidobacterium longum) made from

201 camel milk when compared to cow milk, with inhibitory percentages of 33.2%

202 and 26.4%, respectively, with opposite results observed for the enzyme α-

203 glucosidase (15.5 and 11.3%, respectively).

204 The inhibitory effect of the probiotic dairy foods on the digestive

205 enzymes is dependent on the probiotic strain. The probiotic strains commonly

206 studied were L. casei (Wang et al., 2019; Grom et al., 2020), L. plantarum (Al-

207 Dhaheri et al., 2017; Ayyash et al., 2018, Balthazar et al., 2019), L. brevis

208 (Mushtaq et al, 2019), L. reuteri (Ayyash et al., 2018), L. acidophilus

209 (Apostolidis et al., 2007; Wihansah et al., 2018), L. fermentum (Kinariwala et

210 al., 2019), Enterococcus faecalis (Graham et al., 2019), Bifidobacterium

211 adolescentis (Su et al., 2018), Bifidobacterium bifidum, Bifidobacterium

9
212 infantis, and Bifidobacterium longum (Shori & Baba, 2013). Ayyash et al. (2018)

213 observed that milk fermented with Lb. plantarum K772 strains had higher α-

214 glucosidase inhibitory activity (≈40-60%) when compared with milk fermented

215 by other strains (Lb. reuteri KX881777, Lb. plantarum KX881779, and Lb.

216 plantarum DSM2468, 108 CFU/mL, <35%). Similarly, Graham et al. (2019)

217 observed that milk fermented with E. faecalis DPC5154 demonstrated the

218 highest α-glucosidase inhibitory activity (33.41%) among twenty-five E. faecalis

219 strains studied (15-30%). On the other hand, in Kalahari cheeses, different

220 probiotic strains (Lactobacillus plantarum, NCDC 012; Lactobacillus casei, NCDC

221 297; and Lactobacillus brevis, NCDC 02) exhibited similar inhibitory activities (≈

222 20-45%, Mushtaq et al., 2019). The discrepant results between the types of

223 milk and the probiotic strains may be due to the proteolytic ability of the strains

224 in different dairy matrices, resulting in different effects on the digestive

225 enzymes (Ayyash et al., 2018). In addition, the acidification of products during

226 the fermentation process may affect the functionality, increasing it (Shori &

227 Baba, 2011). Thus, it is essential to evaluate the functionality of the selected

228 probiotic strains in the matrix to be administered.

229 The ability of the probiotic strains to produce exopolysaccharides (EPS) is

230 an important characteristic from a functional point of view, and a greater effect

231 was observed for the use of EPS-forming strains when compared to the use of

232 EPS alone. Low-fat Akawi cheese made with EPS-forming L. plantarum strains

233 (107-108 CFU/g) showed higher α-amylase inhibitory activity (≈ 40-60%, day

234 21) when compared to cheese made with non-EPS-forming strains (≈ 30%, Al-

235 Dhaheri et al., 2017). In addition, the use of EPS-forming probiotic strains

10
236 (Lactobacillus plantarum JLK0142, >7.8 log CFU/g) in the manufacture of low-

237 fat Cheddar cheese was more efficient in inhibiting the α-amylase enzymes

238 (45.63%) when compared to the addition of EPS in its purified form (41.03%,

239 Wang et al., 2019). The better performance of probiotic cultures concerning the

240 production of EPS suggests that the probiotic cultures can act on milk proteins,

241 with greater release of bioactive compounds with antidiabetic activity (Wang et

242 al., 2019). The addition of probiotic cultures can accelerate the proteolysis

243 during ripening of cheeses, enhancing the breakdown of casein into peptides

244 and amino acids, and resulting in a higher concentration of bioactive peptides

245 capable of inhibiting the α-glucosidase and α-amylase enzymes (Wang et al.,

246 2019).

247 The ingredients used in the manufacture of probiotic dairy products have

248 a significant impact on the inhibition of α-glucosidase and α-amylase enzymes.

249 The addition of neem (Azadirachta indica, 11.7% extract), a medicinal plant, to

250 probiotic yogurts (Lactobacillus acidophilus LA-5, Bifidobacterium bifidum Bb-

251 12, Lactobacillus casei LC-01, Lactobacillus rhamnosus, Bifidobacterium longium

252 and Bifidobacterium longium 106 CFU/g) resulted in a higher α-glucosidase (≈

253 6-16%) and α-amylase (44.4%) inhibitory activity when compared to the

254 control yogurt (≈3-15% and 29.8%, respectively, Shori & Baba , 2013). Similar

255 results were observed in several studies, including the addition of kiwifruit-bitter

256 gourd juice (5-15%) to fermented milks containing Bifidobacterium adolescentis

257 (107 CFU/g, Su et al., 2018); 2% soybean oligosaccharides in yogurts made

258 with L. acidophilus (CCFM6) and L. plantarum (CCFM47) (>106 CFU/g, Muganga

259 et al., 2015); and 1% roselle extract in yoghurt from goat milk containing L.

11
260 acidophilus IIA-2B4 (Wihansah et al., 2018). The phenolic compounds present

261 in plants have the ability to bind to the enzyme binding sites, altering their

262 catalytic activity (Shori & Baba, 2013). In addition, soybeans contain isoflavones

263 in the form of aglycones, which have α-glycosidase inhibitory capacity

264 (Muganga et al., 2015). Finally, roselle extract contains phytochemicals, such as

265 anthocyanins, flavonoids, and phenolic compounds, which cause changes in the

266 molecular configuration of the enzymes, besides their hydrophilic and

267 hydrophobic properties, resulting in a decrease in enzyme activities (Wihansah

268 et al., 2018).

269 It is worth mentioning that the effects reported for the plant components

270 were observed in fermented products such as yogurts, suggesting an important

271 role of the fermentation process. As reported by Amirdivani (2015), although

272 garlic (A. sativum) exhibited a low inhibitory activity on α-glucosidase and α-

273 amylase enzymes, a higher inhibitory effect was observed after the addition of

274 garlic (11% in extract form) to probiotic yogurts (108 CFU/ g, Lactobacillus

275 acidophilus LA-5, Bifidobacterium Bb12, Lactobacillus casei LC-01), with IC50

276 values of 28.0 and 302.7 mg/g for α-amylase and α-glucosidase, respectively,

277 when compared to the control yogurt (37.5 and 480 mg/g, respectively). Similar

278 result was observed for kefir made with 15% Aronia juice (Aronia melanocarpa)

279 when compared to the control (IC50 of 152.53 vs 365.16 mg/g, Du & Myracle,

280 2018). Thus, the use of phytochemicals alone may have a little effect on the

281 glycemic control, with a higher functionality in combination with fermented

282 dairy products. The fermentative process and the performance of probiotic

12
283 cultures can promote the release of compounds with antihyperglycemic activity

284 from the plant matrix, contributing to the effects observed (Du & Myracle, 2018).

285 The addition of prebiotic compounds to probiotic dairy products can increase

286 the functionality of the processed products. Balthazar et al. (2019) evaluated the

287 inhibitory effect of the addition of prebiotics (3% inulin, 3% potato starch, or 1.5%

288 of each prebiotics) on α-glucosidase and α-amylase activities in probiotic fermented

289 beverages (109 CFU/g, L. plantarum) made from sheep milk and strawberry pulp. A

290 synergistic effect between prebiotics (inulin and potato starch) and probiotics was

291 observed, and the fermented beverage made with both compounds showed a

292 higher inhibitory activity of α-glucosidase and α-amylase, with values twice as high

293 as those found in beverages with the addition of probiotics + inulin, and probiotics

294 + potato starch, and six times higher than the conventional beverage. Those results

295 indicate that the peptides released due to proteolytic activity of the probiotic culture

296 may have potential functional benefits. Similarly, the addition of prebiotics (inulin or

297 potato starch) increased the proteolytic activity of the microorganism, providing

298 bioactive peptides to functional beverages.

299 The results showed that matured products (ripened cheeses), fermented

300 products, and whey-based products (dairy beverages) have a higher ability to

301 maintain the postprandial blood glucose. In addition, the probiotic strains with

302 higher proteolytic and EPS-forming capacities play an important role in the

303 formation of bioactive compounds capable of inhibiting the α-glucosidase and α-

304 amylase enzymes, with a greater effect on postprandial glycemia. The use of

305 prebiotic components in combination with probiotic cultures with possible

306 synergistic effects is recommended. Finally, the use of plant-derived products may

13
307 improve the effect of the probiotic cultures on dairy products by improving the

308 antihyperglycemic properties.

309 Studies that evaluated the effect of probiotic dairy products and synthetic

310 drugs on digestive enzymes in vitro are still scarce (Wihansah et al., 2018).

311 Wihansah et al. (2018) compared the α-glucosidase inhibitory activity of

312 acarbose and yoghurt made from goat milk containing L. acidophilus IIA-2B4

313 and roselle extract and reported similar results (24.88-33.45 and 25-35 %

314 inhibitory activity at 0.1-0.5 ppm, for acarbose and yogurt, respectively). The

315 results show that, when probiotic cultures and dairy matrices are properly

316 selected, the bioactivity of the probiotic dairy products can reach levels similar

317 to those observed for synthetic drugs. Dairy products naturally functioned as

318 functional foods and were easily included in the diet. This result is potentially

319 important to minimize the side effects of excessively high inhibition of α-

320 amylase, which is observed after the use of synthetic drugs (Ujiroghene et al.,

321 2019).

322 Some authors have proposed strategies to increase the functionality of

323 the probiotic cultures. The encapsulation of a probiotic strain (P. acidilactici)

324 into a novel encapsulating matrix (whey proteins from camel and cow milk)

325 resulted in a 59.32 and 54.82% increase in α-glucosidase inhibitory activity,

326 respectively, after simulated gastrointestinal conditions (Ahmad et al., 2019).

327 The higher inhibitory activity may be due to changes in gene expression profile

328 and stress adaptation of the encapsulated and non-encapsulated probiotic

329 strains. In addition, whey proteins may have degraded to peptides during

330 gastrointestinal transit (Ahmad et al., 2019). Therefore, the use of

14
331 microencapsulated probiotic cultures may result in increased antidiabetic

332 properties in probiotic dairy products.

333 5. Perspectives

334 This is the first review to address the relationship between the

335 consumption of probiotic dairy products and postprandial blood glucose. The

336 results indicate that the type of product, the probiotic strain used, the

337 ingredients incorporated into the product, and the addition of prebiotic

338 compounds to the formulation can directly impact the postprandial blood

339 glucose regulation and/or inhibition of α-glucosidase and α-amylase enzymes.

340 Several studies have focused on in vitro inhibitory effect on digestive enzymes,

341 thus clinical studies are required to investigate the relationship between

342 probiotic products and postprandial blood glucose in humans. The effects can

343 be quantitatively comparable to those provided by synthetic drugs, depending

344 on the dairy matrix and the probiotic strain used, with promising results from a

345 functional point of view, with no side effects. Thus, new probiotic strains with

346 higher proteolytic capacity and/or EPS production ability should be studied in

347 dairy matrices to improve the antihyperglycemic activity.

348

349 References

350 Ahmad, M., Mudgil, P., & Maqsood, S. (2019). Camel whey protein

351 microparticles for safe and efficient delivery of novel camel milk derived

352 probiotics. LWT, 108, 81-88.

353 Al-Dhaheri, A. S., Al-Hemeiri, R., Kizhakkayil, J., Al-Nabulsi, A., Abushelaibi, A.,

354 Shah, N. P., & Ayyash, M. (2017). Health-promoting benefits of low-fat

15
355 akawi cheese made by exopolysaccharide-producing probiotic

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357 Science, 100, 7771-7779.

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495

496

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 Table 1. Probiotic dairy foods and postprandial glycemia studies and/or inhibition of α-glucosidase and α-amylase activities

Dairy food Probiotic strain Study type Results Reference

Prato cheese,
Prato cheese presented: (1) the highest lipid and protein contents,
Minas Frescal In vitro e in
L. casei (2) the highest α-amylase and α-glucosidase inhibition, (3) its Grom et al. (2020)
cheese and Whey vivo (humans)
consumption resulted in a lesser increase in blood glucose level
dairy beverage

Low fat cheeses prepared with EPS-forming L. plantarum strains

Al-Dhaheri et al.
Akawi cheese L. plantarum In vitro showed higher α-amylase inhibitory activity ((≈ 40-60%) than
(2017)
cheeses prepared with non-EPS-forming strains (≈ 30%). There
was no effect on α-glucosidase inhibition.

Lactobacillus plantarum
Kalari cheese with added probiotics exhibited higher α-glucosidase
(NCDC 012),
and α-amylase inhibition activity when compared to the cheese
Lactobacillus casei Mushtaq et al.
Kalari cheese In vitro without added probiotics. Different probiotic strains presented
(NCDC 297), and (2019).
similar inhibition activities (≈ 20-45%)
Lactobacillus brevis
(NCDC 02)
 The use of the EPS-forming probiotic strain in the preparation of
Lactobacillus plantarum low-fat Cheddar cheeses is more efficient in inhibiting α-amylase
Cheddar cheese Ìn vitro Wang et al. (2019)
JLK0142 enzyme (45.63%) than inclusion of only EPS in purified form
(41.03%)

Lb. reuteri-KX881777,
Lb. plantarum-
Fermented milk The fermented milks with strain Lb. plantarum-K772 and those
KX881772, Lb. Ayyash et al.
(bovine and In vitro from bovine milk showed higher α-glucosidase and α-amylase
camel) plantarum-KX881779, inhibitory activities (2018)
and Lb. plantarum
DSM2468

During the fermentation process there is increased α glucosidase

Fermented and
inhibitory activity (>4%) in fermented milks added with L. Apostolidis et al.
non-fermented L. acidophilus In vitro
milk
acidophilus (> 106 cfu/g). (2007)

Inhibition increased during the milk fermentation process (24 h),

with an increase of 59.47% for strain M2 and 53.45% for strain Kinariwala et al.
Fermented milk L. fermentum M2 e M7 In vitro
M7 in α-amylase activity. For α-glucosidase activity, increases of (2019)
7.09 and 8.36% were observed, respectively.
 Enterococcus faecalis Milk fermented with E. faecalis DPC5154 demonstrated the highest Graham et al.
Fermented milk In vitro
(25 strains) level of α-glucosidase inhibitory activity (33.41%). (2019)

Fermented
beverage A synergistic effect was observed between prebiotics (inulin and
manufactured
potato starch) and probiotic culture, and the fermented beverage
with semi-
L. plantarum (CECT that had all the components showed higher inhibitory activity of α- Balthazar et al.
skimmed sheep In vitro
milk and 8328) glucosidase and α-amylase, with mean values twice as those found (2019)
strawberry pulp for probiotic + inulin and probiotic + potato starch beverages, and
and added six times higher than those of conventional beverages.
prebiotics

Fermented milk The addition of Kiwifruit-Bitter gourd juice resulted in higher α-

Bifidobacterium glucosidase (21.6%) and α-amylase (29.57%) inhibitory activity
with Kiwifruit- In vitro Su et al. (2018)
adolescentis than the conventional fermented milk (21.6 and 25.53%,
Bitter gourd juice
respectively).
 Lactobacillus
acidophilus LA-
5, Bifidobacterium
bifidum Bb-
Yogurt with neem 12, Lactobacillus
The addition of neem (Azadirachta indica) resulted in higher α- Shori & Baba
(Azadirachta casei LC-01, In vitro
glucosidase and α-amylase inhibitory activity. (2013)
indica) Lactobacillus
rhamnosus, Bifidobacter
ium
infantis and Bifidobacter
ium longum
Lactobacillus
acidophilus LA-
5, Bifidobacterium
bifidum Bb-
The inhibition of α-amylase in yogurts was stronger in camel-milk
Yogurt with 12, Lactobacillus
yogurt (33.2%) than cow-milk yogurt (26.4%). The reverse was Shori & Baba
Cinnamomum casei LC-01, In vitro
true for α -glucosidase (15.5 vs 11.3%). The addition of (2011)
verum Lactobacillus
Cinnamomum verum improved the inhibitory activity.
rhamnosus, Bifidobacter
ium
infantis and Bifidobacter
ium longum

Yogurt with
L. acidophilus(CCFM6)
soybean SBOs enriched yoghurt improved α -glucosidase inhibition (>25 vs Muganga et al.
and L. In vitro
oligosaccharides 9%, P<0.05). (2015)
(SBOs) plantarum(CCFM47)
 Garlic (A. sativum) has low inhibitory activity of the α-glucosidase
and α-amylase enzymes. However, when garlic (11% as an
Lactobacillus acidophilus
extract) was added to probiotic yogurts, there was an increase in
Yogurt with garlic LA-5, Bifidobacterium
In vitro enzyme inhibitory activity (IC50 of 28.0 mg/g for α-amylase, and Amirdivani (2015)
(Allium sativum) Bb12, Lactobacillus
302.7 mg/g for α-glucosidase) when compared to control yogurt
casei LC-01
(IC50 of 37.5 mg/g for α-amylase, and 480 mg/g for α -
glucosidase).

Goat milk yogurt Roselle extract enriched yoghurt improved α-glucosidase inhibition
Wihansah et al.
with roselle L. acidophilus IIA-2B4 In vitro (35 vs 15%, P<0.05). Inhibition was comparable with acarbose at
(2018)
extract 0.1-0.5 ppm concentration (24.88-33.45%).

Kefir with Aronia

Aronia juice enriched yoghurt improved α-glucosidase inhibition Du & Myracle
juice (Aronia Diverse In vitro
(IC50 of 152.53 vs 365.16 mg/g). (2018)
melanocarpa)
Figure 1. Mechanism of action of probiotic cultures in the inhibition of α-amylase

and α-glucosidase enzymes. Adapted from Patil et al. (2015).

1 Probiotic dairy foods and postprandial glycemia;

2 Mechanisms of action of probiotics are presented;

3 Effect of dairy matrix should be considered.

4
5
6
7