1. Introduction
Zn is an essential trace element required for maintaining the normal growth and development of the human body. A human body contains 2–3 g of Zn, approximately 90% of which is found in muscles and bones [
1]. Healthy adults consume approximately 10–15 mg of Zn daily through their diet, with a general absorption rate of 20–30%. Zn deficiency is a common nutritional deficiency worldwide, affecting about 31% of the global population, particularly in developing countries [
2]. Notably, mild-to-moderate Zn deficiency is widespread [
3]. Especially young children, as well as pregnant and postpartum women, are at a high risk of Zn deficiency [
4]. Growth retardation and hypogonadism are known to result from a lack of Zn [
5]. According to Fons et al. [
6], 25 children with low serum Zn levels were characterized by substantially lower values of bone age delay, growth velocity in mm/month, and ratio between calculated and theoretical growth velocity for bone age. Zn supplementation during the right periods throughout development may also help to restore growth and development. After three months of Zn treatment, 50 pre-pubertal Egyptian children with low stature and Zn deficiency exhibited substantial increases in height standard deviation scores (SDS,
p = 0.033) [
7]. Furthermore, it has recently been identified that Zn is critical for male fertility, and that Zn shortage causes sperm abnormalities, as well as a decrease in serum testosterone concentration [
1].
At present, Zn supplementation for Zn deficiency is mainly achieved by additional intake of Zn supplements, including inorganic Zn (e.g., ZnO, ZnSO
4, and ZnCl
2), simple organic Zn (e.g., Zn gluconate, Zn acetate, and Zn propionate), and organic Zn (e.g., amino acid-chelated Zn and protein-complexed Zn). However, the absorption efficiency varies significantly among the different Zn supplements [
8,
9]. Organic Zn is more easily absorbed by the human body than inorganic Zn, and the inorganic Zn is usually accompanied by side effects. Compared with the organic Zn, the simple organic Zn has certain side effects, such as irritation of the gastrointestinal tract. Organic Zn is mainly synthesized artificially, which is relatively safe, but its synthesis is complicated [
10]. The enrichment of Zn by microorganisms has been extensively studied in recent decades [
11]. Zn-enriched bacteria are produced during their growth on a Zn-containing medium [
12] and the inorganic Zn can be converted into organic Zn, which is more easily absorbed and utilized. Probiotics are a kind of living microorganism, beneficial to the health of the host [
13,
14], and can be used in the field of Zn enrichment. In addition, the diversity and the stability of the intestinal microbiome will decrease significantly in the case of long-term Zn deficiency [
15,
16]. Zn-enriched probiotics can also regulate the intestinal microbiome [
17] besides supplementing Zn. However, it remains unknown whether changes in the gut microbiota have an impact on the growth and reproduction of the host. As a new type of dietary Zn source, Zn-enriched probiotics have more advantages than other Zn supplements and are, thus, worth exploring deeply.
Given the presumed advantages of using Zn-enriched bacteria, we investigated the bioavailability of three different Zn supplements; that is, ZnO, Bifidobacterium longum + ZnO, and Zn-enriched B. longum, upon which Zn intake and the effects on growth and reproductive development of rats were examined. Using a Zn deficiency rat model, the effects of low, medium, and high doses of Zn supplementation on growth, reproductive development, Zn concentration in tissues, blood biochemistry, and the gut microbiota were assessed in rat pups. This study lays the foundations for the development of Zn supplements with high absorption efficiency and no side effects.
4. Discussion
Zn supplementation could improve the growth and reproductive development of Zn-deficient rat pups [
34,
35], despite no significant variations in body weight and testicular weight between intervention groups. We still considered them crucial, particularly due to the importance of internal factors. In contrast, body and testicular weights in the high dose group of Zn-enriched CCFM1195 were slightly decreased. We speculated that the Zn levels in the body were higher due to higher bioavailability, which increased the burden of metabolizing this micronutrient. While the liver is the main organ involved in Zn metabolism, the small intestine, liver, and pancreas maintain Zn homeostasis [
36]. It has been documented that excessive Zn will reduce the weight of the pancreas [
37]. In addition, an excess of Zn will compete with other micronutrients [
38], affecting the absorption of copper, iron, calcium, and other elements, which will also have adverse effects on the body.
The inner connection caused by different Zn supplements at different doses will inevitably lead to different levels of Zn in the serum and organs. Zn absorbed by the intestine first enters the blood, combines with albumin in the blood (above 94%), and is then transported to the liver (about 67–80% of total Zn absorbed) [
39]. The utilization ratio of the Zn absorbed, as evaluated by serum Zn levels and Zn deposits in the liver, was highest for Zn-enriched CCFM1195, less for CCFM1195 + ZnO, and least for ZnO. The trend was even more pronounced at low Zn supplementation. This result also verified our previous speculation on the Zn absorption efficiency associated with the three Zn supplements. With respect to a normal diet, many factors affect the bioavailability of Zn, such as phytic acid, high dietary fiber, and polyphenols, which form insoluble complexes with Zn [
40], reducing the bioavailability of exogenous Zn and decreasing the absorption of Zn in the small intestine. In this case, most of the exogenous Zn will be transported to the colon to compensate for the impaired absorption of Zn in the proximal region [
41]. In addition, other metal ions, such as Ca
2+ or Fe
2+, compete with Zn
2+ for intestinal absorption [
38]. Zn-enriched probiotics chelate Zn with suitable complexing strength. Because trace elements are protected by the ligand after entering the digestive tract, this approach can help avoid the influence of physical and chemical factors that are not conducive to metal absorption in the gastrointestinal tract, reduce the antagonism among different metal ions, and the negative effects of phytate on the absorption of Zn in the small intestine [
8,
42], thereby increasing the absorption rate. Short-chain fatty acids (SCFAs) are produced by bacterial fermentation and serve as the main metabolic substrate of colon cells [
43]. As such, SCFAs can increase dietary Zn absorption by decreasing the pH in the intestinal lumen [
44], thus, increasing the solubility of Zn, or by stimulating the proliferation of intestinal epithelial cells to increase the total absorption area of the intestine [
45]. Furthermore, studies have shown in recent years that some probiotics (mainly
Lactobacillus and
Bifidobacterium) can degrade oxalate in the intestine [
46,
47,
48,
49,
50]. Together, these studies may help explain why the bioavailability of Zn-enriched CCFM1195 and CCFM1195 + ZnO was higher than that of ZnO alone. However, why the bioavailability of Zn-enriched CCFM1195 was in turn higher than that of CCFM1195 + ZnO is not yet known; it may be dependent on the ratio of organic to inorganic Zn, which must be confirmed by further studies. At present, we have proven that the proportion of organic Zn in Zn-enriched CCFM1195 is much higher than that in CCFM1195 + ZnO and that their morphologies differ. The organic Zn of Zn-enriched CCFM1195 comprises mainly Zn bound to intracellular molecules, whereas that of CCFM1195 mixed with ZnO comprises mainly Zn bound to the cell wall.
The different Zn levels in the body, in turn, generated activity differences of Zn-dependent enzymes and changes in hormone levels. Ultimately, growth and reproductive development will be affected. Different Zn supplements had different abilities to restore ALP enzyme activity. Our results indicated that Zn-enriched CCFM1195 had the strongest ability to restore enzyme activity, followed by CCFM1195 + ZnO, while ZnO was the worst, which was in line with previously obtained results. In terms of the GH and testosterone concentrations in the serum, our results showed that the ability of Zn-enriched CCFM1195 to increase the GH concentration was most significant. Additional Zn supplementation improved the testosterone concentration as a function of the dose administered, but no significant difference in testosterone concentration was observed compared to the Zn-deficient group. However, additional Zn supplementation resulted in significant differences in testis weight and testicular Zn content, indicating that Zn may act on targets downstream of testosterone to regulate reproductive development.
The cause of all these differences may have risen from the difference in absorption and utilization of the different Zn supplements at different doses by the body. The Zn content in the ileum, cecum, colon, and feces was measured. Zn is mainly absorbed in the small intestine, with very little absorption in the stomach and an auxiliary effect within the large intestine [
51]. The accumulation of Zn in each intestinal segment can be considered indicative of the state of Zn to be absorbed. In general, ZnO led to the highest accumulation rates in the three intestinal segments, followed by CCFM1195 + ZnO, while Zn-enriched CCFM1195 was associated with the lowest accumulation rates. Unabsorbed dietary Zn (exogenous Zn) and endogenous Zn produced from pancreatic and biliary secretions, gastroduodenal secretions, transepithelial flow from enterocytes or other intestinal cell types, and mucosal cell sloughing, were all excreted in the feces [
36,
52]. There is a Zn homeostasis regulation mechanism in animals, and Zn absorption and endogenous secretion are the main ways of Zn homeostasis regulation [
53]. The gastrointestinal system, particularly the small intestine, liver, and pancreas, is thought to play a major role in maintaining Zn homeostasis [
52]. The maintenance of Zn metabolism in the body mainly depends on the regulation of intestinal excretion. When the body takes in too much Zn, the amount of Zn secreted into the small intestine by the liver and pancreas increases, thus, increasing endogenous Zn exclusion to achieve a regulation of Zn supply. Zn is released from the body into the colon without being absorbed, resulting in endogenous Zn loss via the feces. The major source of endogenous Zn re-excretion into the intestinal lumen is pancreatic production (which is six times higher than secretion through bile) [
54]. Researchers have reported that an enteropancreatic circulation exists in humans. Much of this Zn must eventually be resorbed to avoid a negative Zn balance. According to the Zn content in feces collected after 24 h of gavage, ZnO could not stay in the intestinal tract for a long time and was excreted soon after being eaten, resulting in the lowest fecal Zn content in the ZnO groups. Zn-enriched CCFM1195 could stay in the gut for a long time. Compared with the ZnO groups,
B. longum CCFM1195 could increase the residence time of ZnO in the intestine, which may have increased the total amount of ZnO absorbed and promoted the absorption process. Therefore, we speculated that Zn was more efficiently absorbed when administered as Zn-enriched CCFM1195 than when given as ZnO. CCFM1195 + ZnO could be placed between the other two Zn supplements in terms of absorption efficiency.
Pharmacokinetic data analyses confirmed our previous speculation on the absorption of ZnO, CCFM1195 + ZnO, and Zn-enriched CCFM1195. The absorption and metabolism pathways of inorganic and organic Zn are different. Inorganic Zn was not readily absorbed, and only when transformed into organic Zn could be absorbed, transported, and utilized by the body. Organic Zn existed stably in the digestive tract and was absorbed by the brush-like border of the small intestinal villi, in the form of amino acids or peptides. It did not form complexes with cellulose and phytic acid, which are difficult to be absorbed and utilized [
42]. Moreover, antagonistic effects between elements were also avoided. ZnO was quickly excreted through absorption and metabolism cycles in the body and its bioavailability was poor. After being enriched by bacteria, inorganic Zn existed in the digestive tract in an organic form, combined with cellular structures [
11]. They were slowly released and absorbed, following bacterial death in the intestine, so that the body could maintain higher Zn levels for an extended period of time than when supplemented with inorganic Zn.
Species diversity and abundance of the gut microbiota in rat pups were examined using 16S rRNA sequencing. The results showed that the diet was the main contributor to the gut microbiota profile, followed by the litter effect. The impact of Zn supplementation alone on the overall composition and diversity of the gut microbiota was minor, as indicated by the lack of substantial changes in gut microbiota species after ZnO therapy. Accordingly,
B. longum CCFM1195 increased the microbial abundance and diversity within the samples, except for the high dose of Zn-enriched CCFM1195. The LEfSe analysis revealed that
Lactobacillus and
Blautia dominated the microbiota of the control group at the genus level, at 24.63% and 21.32%, respectively. In the model group,
Blautia,
Ruminococcus gauvreauii,
Flavonifractor, and
Negativibacillus were decreased, while the
Lachnospiraceae NK4A136 group had increased to become the dominant genus.
Blautia had a special anti-inflammatory effect, and the decrease in its abundance may have led to the loss of anti-inflammatory effects [
55]. During Zn treatment,
Blautia was found to be the dominant genus. With the administration of the Zn dose, the proportion of
Lactobacillus had increased, while
Blautia had decreased. Zn supplementation was helpful to maintain the stability and diversity of the intestinal microflora. The intestinal microbiota of
Firmicutes had increased, especially
Lactobacillus [
56]. Moreover, the two genera were strongly out of proportion in the ZnO groups. In the CCFM1195 + ZnO-H group, the ratio of these two genera was nearly re-balanced. In the case of medium and high doses of Zn-enriched CCFM1195, the relative abundance of
Lactobacillus exceeded
Blautia, representing the dominant genus. Probiotics, such as
Lactobacillus,
Bifidobacterium, and
Enterococcus, can improve the host’s gut microbiome balance and help restore or maintain a beneficial gut microbiome to prevent digestive disorders and potentially help growth and development [
57]. Although Zn supplementation is evidently associated with specific changes in the gut microbiota, the relationship between the dynamic changes of the microbiota and Zn supplementation is not entirely established. Further research is required to evaluate the role of the gut microbiota in the process of Zn deficiency supplementation.