Snacks are considered healthy if they provide a balanced amount of energy and essential nutrients to meet nutritional needs. Dairy products are a good base for the production of snacks and a great food source of calcium for the prevention of osteoporosis, especially for the elderly. For this purpose, dairy snack formulations were designed to improve calcium bioaccessibility, which was measured by a three-step in vitro method to simulate oral, gastric and intestinal digestion. Calcium bioaccessibility was assessed as the percentage of soluble calcium under intestinal digestion in relation to total calcium. The changes in calcium speciation (i.e., ionic calcium and soluble calcium) during in vitro static digestion were followed by combining electrochemical determination of ionic calcium with inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis of total calcium and soluble calcium.
The yogurt snacks rich in calcium (2.8-7.4 mg/g snack) were formulated with milk proteins, high methoxyl pectin and sucrose, and freeze-dried to produce crispy snacks with a yogurt flavor. Pectin addition was found important to provide snacks with a suitable texture, considering crispness and hardness. The ionic calcium decreased by 70% from the gastric (pH 3.0) to the intestinal (pH 7.0) phases, and continued to decrease during intestinal digestion. The decrease of soluble calcium indicates a competition between absorption and precipitation from the beginning to the end of intestinal digestion. The addition of whey protein isolates provided snacks with the highest calcium bioaccessibility of 27% among the designed snacks at the end of intestinal digestion, despite the lowest initial total calcium content. The presence of micellar caseins resulted in yogurt snacks with the lowest calcium bioaccessibility, even though the total calcium content was higher. Heat treatment of yogurt bases at 90 ℃ prior to freeze-drying and the addition of high methoxyl pectin had little effect on the calcium bioaccessibility for all types of snacks.
Whey mineral concentrates, a by-product in cheese production containing insoluble calcium phosphate, increased the calcium bioaccessibility in snacks when added to the yogurt formulations in combination with lime juice. For the snacks without lime juice, calcium bioaccessibility at the end of intestinal digestion was between 17% and 25%. For the snacks containing lime juice, calcium bioaccessibility increased to between 24% and 40% due to the presence of citric acid in lime juice. Citric acid enhanced the calcium solubility both in whey mineral concentrates and yogurt keeping calcium supersaturated under neutral conditions, although the complex binding reduced the amount of ionic calcium. The combination with whey protein isolates or whey protein hydrolysates further improved calcium bioaccessibility in snacks at the end of digestion, more significantly for whey protein isolates.
The effect of pectins with different degrees of esterification (DE) on calcium bioaccessibility in yogurt snacks was quantified, combined with or without whey mineral concentrates solubilized by lime juice. Pectin improved the texture of the snacks but lowered the calcium bioaccessibility by 6%-16% (except for DE70) through the binding to carboxylate groups of pectin. Decreasing DE of pectin increased calcium binding and reduced calcium bioaccessibility, as indicated by the estimated binding sites and association constants calculated by isothermal titration calorimetry. At the same time, the increase of temperature from 25 ℃ to 37 ℃ and pH from 4 to 7 promoted the endothermic interactions between calcium and pectin. Calcium binding to low methoxyl pectin was found to compete with complex binding to citrates from the lime juice, resulting in decreased calcium bioaccessibility in the intestines. High methoxyl pectin with a lower binding affinity for calcium had little effect on calcium bioaccessibility.
To better understand the increased calcium bioaccessibility provided by whey proteins, the in vitro digestion of the two major whey proteins, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), in the presence of calcium chloride was investigated. α-La and, more significantly, β-Lg were found to increase calcium bioaccessibility during intestinal digestion as calcium complex bound to peptides formed from the protein hydrolysis by gastrointestinal enzymes. The amount of complex bound calcium in the presence of β-Lg was almost twice as high as that of α-La during digestion. Based on LC-MS/MS analysis and visualization of amino acid sequences, peptides containing aspartic acid and glutamic acid acting as calcium chelators are suggested to prevent calcium precipitation in the intestines.
Crispy yogurt snacks with high calcium bioaccessibility were obtained by freeze-drying using a yogurt formulation consisting of whey protein isolates that increased protein content, whey mineral concentrates solubilized by lime juice increasing calcium bioaccessibility, and calcium-insensitive high methoxyl pectin that improved textural properties. The results of this study provide knowledge to support the development of healthy snacks with increased calcium bioaccessibility.