1. Introduction
The botanical kingdom offers endless opportunities to isolate new functional substances, such as those originating from various berries, like the Goji berry [
1,
2]. These substances may consist of polyunsaturated fatty acids, flavanols (anthocyanins, proanthocyanidins and quercetin) and ellagic acid, which exhibit antioxidant, antimicrobial, anti-inflammatory and anti-mutagenic properties [
1,
3,
4,
5,
6].
L. barbarum (Goji berry) is of great importance as a functional food product and has been used for more than 2500 years for nutritional and medicinal purposes [
7].
L. barbarum, is a solanaceous defoliated shrubbery native to Asian areas such as China and Tibet [
8]. It is popular in traditional medicine in Asian countries due to its biofunctionality, which is related to various components that include polysaccharides, carotenoids, and phenolic compounds [
9]. The ancient herbalist classics recorded that
L. barbarum nourishes the liver and kidneys and brightens the eyes [
9]. Recent studies have shown that
L. barbarum exhibits neuroprotective and hepatoprotective properties, potential benefits against cardiovascular and inflammatory diseases, anti-diabetic action by improved control of glucose metabolism, anti-aging effects, cytoprotection and protection from memory deficiencies and lung disorders, and anticancer activity [
10,
11,
12], thanks to which,
L. barbarum fruit has recently gained increasing popularity in Europe and North America [
12].
Recently Goji berries have been also positively evaluated for their prebiotic potential in foods like yogurt [
13], since their polysaccharides may be selectively utilized by some probiotic bacteria [
14], although a potential prebiotic effect may be also linked to other molecules like polyphenols, which may stimulate the growth of probiotic bacteria in the gut, or inhibit the growth of antagonistic bacteria in the complex intestinal microbiota [
15].
Gut colonization by beneficial probiotic bacteria is recognized as an essential parameter for intestinal health, and human health in general. It occurs in early life, as
Bifidobacterium and
Lactobacillus species attach to the gastrointestinal tract, which is necessary for establishing the gut mucosal barrier, maturation and modulation of the immune system, preventing infections by enteric pathogens and improving gastrointestinal function, digestion, and metabolism [
16,
17,
18,
19,
20]. Nowadays, the presence (or supplementation) of certain probiotic bacteria, prebiotics or symbiotics (mixed preparations of probiotics and prebiotics) in the gastrointestinal tract is linked to prevention or reduced risk of ulcer, gastroenteritis, inflammation, colon cancer and metabolic syndrome (the latter involving hypolipidemic, hypocholesterolemic and potential hypoglycemic activity) as well as preterm birth and neonatal gastrointestinal disorder [
21,
22,
23,
24,
25,
26,
27,
28,
29]. However, gut microbiota are not stable throughout life, and significant changes can occur naturally throughout the life cycle, or be provoked by many factors, including a diet poor in plant fiber and prebiotics. Thus, foods that are rich in prebiotics (i.e., carbohydrates or other components that can be selectively metabolized by probiotic bacteria and alter the gastrointestinal microbiota in favor of the probiotic bacteria), are of great importance in the modern diet [
18,
30,
31,
32], although the effects of prebiotics, even in high doses, cannot be surpassed by a healthy, balanced diet [
30]. Thus, novel probiotics, prebiotics, and synbiotics from different sources have been constantly sought after in recent decades, in order to form part of a fortified functional food for infants, children, or adults, or a food supplement for nutraceutical and therapeutic purposes [
33,
34,
35,
36]. Notably, the action of probiotics in the gut is generally shorter, in comparison to the action of prebiotics which can be traced for several weeks after their administration, and also, the administration of exogenous probiotics is usually a short-term treatment (e.g., after antibiotics administration) [
32]. Therefore, the characterization and consumption of novel prebiotics as part of a healthy diet is of great importance, in order to induce the endogenous health-promoting gut microbiota in the long term.
In this respect, the aim of this research was to investigate the in vitro prebiotic potential activity of Goji berry fruit extracts with regard to potential stimulatory effect on the growth of probiotic bacteria (i.e., Lactobacillus and Bifidobacterium species) in nutritive synthetic substrate. Furthermore, the prophylactic effect towards probiotic bacteria in simulated gastric and intestinal juices was studied.
3. Results and Discussion
The data of culture pH and bacterial growth (log cfu/mL) in the synthetic growth medium were collected at 8, 24 and 34 h after the initial inoculation (
Table 2 and
Table 3, respectively). The results shown in
Table 2 suggest that
L. acidophilus exhibits the highest lactate-producing activity, which was evident in the greatest pH reduction (from 6.5 to 3.63 after 34 h of incubation) among the five probiotic bacteria tested with or without encapsulated Goji berry extract addition. Also, lactate production and consequent pH reduction appears to be enhanced after the addition of sample 3 to
L. acidophilus cultures, especially at 24 and 34 h (lower pH compared to the control by 0.52 and 0.20, respectively), and to lesser extent, after the addition of sample 1 (pH reduction by 0.5 compared to the control, at 8 h).
L. casei and
L. rhamnosus exhibited a similar trend of significant reduction of culture pH compared to the control, especially after the addition of samples 3 and 1, while the addition of sample 2 had a minor (not statistically significant) effect on stimulating lactate production in
L. casei and
L. acidophilus.
With regard to the two tested
Bifidobacterium species, the addition of all Goji berry extract samples (1–3) led to a slight pH reduction in both species, but
B. longum cultures seemed to produce significantly more organic acids at 24 and 34 h after the addition of Goji berry extracts, compared to the control, especially when sample No 3 was added to the culture, which resulted in a pH drop by 0.34 at 34 h compared to the control (
Table 2). Organic acid production and pH reduction of the culture medium was only stimulated in
B. lactis after the supplementation of sample 3. Overall, it appears that sample 3, which was the Goji berry extract with the highest amount of polysaccharides, as well as polyphenols, had the highest stimulatory effect upon organic acid production in all probiotic
Lactobacillus and
Bifidobacterium species tested (
Table 2).
Apart from increased acid producing capacity, the (higher) reduction of pH of a probiotic culture may also indicate or correlate with an increased population. Indeed, by adding sample No 3 to
L. casei, which had the highest population at 34 h among the 3
Lactobacillus species, a significant increase in the population was observed after 34 h of incubation, reaching a maximum difference of 1.34 log cfu/mL compared to the control at 34 h (
Table 3). Samples 1 and 2 also enhanced the growth of this species at 34 h. A similar (but less pronounced) stimulation of growth at 34 h was evident in
L. acidophilus cultures after the addition of samples 1, 2, and 3 (
Table 3). In fact, it seems that the addition of the Goji berry extracts prolongs the growth or stationary phase of these two
Lactobacillus species, compared to the control, which enters the death phase earlier (before 34 h), since the latter (control) has a significantly reduced population at 34 h compared to 24 h. On the other hand, the growth of
L. rhamnosus was not significantly affected by the presence of Goji berry extracts, which only caused a minor (not significant) increase in the population at 24 h after the addition of samples 3, 1, and 2 (
Table 2).
Between the two along
Bifidobacterium species
B. lactis showed the highest stimulation of growth after addition of the three Goji berry extracts, having 2.0, 1.8, and 1.75 log cfu/mL higher population compared to the control at 34 h, after the addition of samples 3, 2, and 1 respectively (
Table 3). The growth of
B. longum was less affected but was mainly favored by sample 3, at 34 h (increased by 0.26 log cfu/mL) and to a lesser extent by sample 1 (
Table 3). These results indicate a potential stimulatory effect of the encapsulated Goji berry extracts, and especially sample 3, on the growth and viability of most
Lactobacillus and
Bifidobacterium tested here, which is crucial in exhibiting their probiotic properties.
According to Pavli et al. [
40], in order for probiotic bacteria to provide their benefits to consumer’s health, they should remain viable during their transition to the gastrointestinal tract at population levels of 10
6–10
7 cfu/g. Some of the most important inhibitory factors against probiotic bacteria are the acidic environment and the effect of the proteolytic enzyme pepsin in the stomach. According to Ruiz et al. [
41], the bacteriocidal activity of bile salts is another important factor that affects probiotic bacteria in the intestinal environment. In order to test further the viability and survival of probiotic strains in simulated gastric and intestinal conditions, and the potential protective effect of Goji berry extracts in those environments, the two
Bifidobacterium species, along with
L. casei (which had the highest stimulatory effect on growth after addition of Goji berry extract, among the three
Lactobacillus species) were used.
In our study,
L. casei survived well in a simulated gastric juice and was positively affected by the added Goji berry extracts, in a similar as
B. lactis, while
B. longum was less resistant to prolonged acidic conditions and action of pepsin in gastric juices, although it was favored greatly by the addition of Goji berry extracts (
Table 4). More specifically, the control of
B. longum experienced a sheer reduction from 8.84 to 4.7 log cfu/mL after 3 h in gastric juice, while addition of sample 3 preserved the viable population at 7.02 (2.32 log cfu/mL higher than the control) and samples 1 and 2 resulted in a lower, but still important improvement of survival after 3 h in gastric juice by 0.6 log cfu/mL (
Table 4).
B. lactis also benefited from the presence of samples 3, 1, and 2 in the gastric juice, since it had a higher survival compared to the control at 34 h, by 0.98, 0.67, and 0.63 log cfu/mL, respectively.
L. casei population was only slightly increased by the addition of Goji berry extracts (by 0.18 and 0.16 log cfu/mL after the addition of samples 1 and 3, respectively), following 3 h of incubation (
Table 4).
With regard to the survival in intestinal juice,
B. longum exhibited the best survival under these conditions and the optimal protective effect of Goji berry extracts, especially sample 3, after 1 and 3 h of incubation, when it attained an increased population by 0.94 and 1.14 log cfu/mL compared to the control (
Table 5). Samples 1 and 2 were also beneficial for its survival but to a lesser extent. Similarly,
B. lactis retained a higher population by ~0.8 log cfu/mL at 1 and 3 h of incubation in intestinal juice supplemented with sample3, while sample 1 and 2 had a less important protective effect at 3 h (
Table 5).
L. casei was severely affected by the bile salts of the intestinal juices at 1h and especially at 3h of incubation. Even though the population fell below the detection limit at 3 h, there was a significant protective effect in cell viability at 1 h, after the supplementation of samples 3, 1, and 2 (with a decreasing order of significance), showing that Goji berry extracts also affect the survival of
L. casei in intestinal juices (
Table 5). The better tolerance of
Bifidobacteria in the simulated intestinal environment compared to
Lactobacilli could be due to a better capacity of
Bifidobacteria to produce fibrils of exopolysaccharides, which have a protective effect, as well as a role in the adhesion to epithelial cells [
42].
Despite the better survival of probiotic bacteria, especially
Bifidobacteria and
Lactobacilli, has been previously recorded as a result of the addition of Goji berry [
13,
43,
44,
45], the exact mechanism of stimulation or prophylactic action in gastric or intestinal juices is not thoroughly understood, and the distinct physicochemical properties of the foods/substrates in which Goji berry was previously added as a prebiotic, such as yogurt [
43], Chinese pickles [
44], and Goji berry juice [
45] do not allow a clear answer to this question, as many factors such as protein and lipid content, microbial antagonism, etc. may also play a role in the growth and survival of probiotics. In our study, it appears that the concentration of Goji berry polysaccharides plays a significant role and is positively related to the stimulatory of prophylactic effect under stress conditions. More specifically, sample 3, which has the lowest amount of maltodextrin and highest amount of Goji berry extract and polysaccharide content, had the best overall prebiotic activity in both growth media and in gastric and intestinal juices, followed by sample 1, which had the second highest polysaccharide content. The beneficial effect of polysaccharides of
Lycium barbarum and
Astragalus membranaceus herb extracts in rats with acetic acid (AA)-induced ulcerative colitis have been previously documented by Zhao et al. [
46], who linked the prophylactic effects of the plant extracts with a higher proliferation of epithelial cells and the promotion of intestinal barrier recovery after damage in vitro, as well as reduced mucosal damage, weight loss, and diarrhea in vivo. This therapeutic action may also involve improved survival and colonization of the gut by
Bifidobacteria, and to a lesser extent
Lactobacilli, in the presence of Goji berry polysaccharides. Furthermore, it is known that different monosaccharides from Goji berry polysaccharides can be used by lactic acid bacteria (LAB) [
14] and thus enhance the production of lactic acid and other organic acids, resulting in the reduction of pH in the intestinal tissue. This also inhibits the growth of pathogenic microorganisms in the gut, causing significant structural cell membrane damage, protein denaturation, and the loss of enzyme functionality [
47]. Previous studies have investigated the effect of various oligosaccharides, polysaccharides (including maltodextrin), and dietary fibers on the development of probiotic microorganisms, as well as on symbiotic formulations [
48]. Kimoto-Nira et al. [
48] suggest that the presence of certain sugars in the growth medium of probiotic bacteria positively affect their development, and thus probiotic action. According to the results presented in our study, the addition of the encapsulated sample 3 in the cultivation of
L. acidophilus after 24 h caused a reduction of pH by ~0.5 compared with the control. However, pH reduction was not followed by a significant increase in the bacterial population. The possible explanation of this observation is that even if the bacterial population remains fairly stable, lactate production may be induced by the addition of encapsulated Goji berry extracts.
Vernazza et al. [
49] showed that maltodextrin has limited effect on acid production and growth rate (h
−1) of
B. Longum Bb-46, whereas galactooligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharide (XOS), and glucose can increase the growth rate by 0.066, 0.027, 0.1040, and 0.0995 h
−1, respectively. Also, a probiotic
L. plantarum species was shown to produce glucomannan exopolysaccharides, utilizing different sugars, such as lactose, glucose, galactose, and sucrose [
50]. Notably, a large concentration of galactose, as well as fructose and xylose oligosaccharides, are present in Goji berry polysaccharides [
51,
52], which may stimulate exopolysaccharide synthesis by probiotic bacteria and explain the stimulatory effect of Goji berry polysaccharides observed in our study. More specifically, the survival and prevalence of probiotic bacteria in the gastrointestinal tract is partly dependent on the existence of extracellular polysaccharides produced by certain LAB as well as by
Bifidobacteria [
41,
51]. These polysaccharide capsules act as a “shield”, reducing the contact of the cells with the outer environment and facilitating survival under environmental stress conditions [
42,
51]. Interestingly, many of the building blocks of Goji berry polysaccharides, such as galactose, glucose, and mannose among others, are also components of the protective extracellular capsule that probiotic bacteria can synthesize [
53]. Among 21 probiotic strains of
Bifidobacteria (belonging to three different species) and Lactobacilli (belonging to four different species) isolated from human intestinal microbiota, it was found that the produced exopolysaccharides contained galactose and glucose in all biopolymers, and rhamnose in half of them, while mannose, fucose, and
N-acetyl-glucosamine were also minor components in four
Bifidobacterium and one
Lactobacillus strains [
52]. Therefore, it can be deduced that the utilization of Goji berry sugar moieties, and especially galactose, which is in high concentration both in Goji berry and probiotic bacteria polysaccharides, for building a protective exopolysaccharide layer in probiotic bacteria, may also be an alternative mechanism of prebiotic activity of Goji berry polysaccharides.
Also, the results of Giavasis et al. [
54] showed that plant polyphenols (such as olive polyphenols) can have significant stimulatory effects upon the growth and lactate production of certain LAB, whether the polyphenols are encapsulated in maltodextrin or not. The results of the present study confirm these findings and the fact that maltodextrin is not an active prebiotic substance, since sample No 3 with only 2% maltodextrin was more efficient as prebiotic, compared to sample 2 (with 14% maltodextrin) and No 1 (with 7% maltodextrin). Also, the final encapsulated powder of sample 3 had the highest content of total polyphenols, as well total polysaccharides, (followed by sample 2, which had the second-best prebiotic effect), to which the comparatively better prebiotic activity could be attributed to. In fact, other studies have also shown that plant polyphenols can act (at low concentrations) as stimulants of LAB or probiotic bacteria in food (e.g., yogurt) [
55,
56] or in vivo [
57]. Kafantaris et al. [
57] reported that, when piglets were treated with grape pomace polyphenols, they had significantly lower levels of Enterobacteriaceae in their feces, and significantly increased levels of
Bifidobacterium and
Lactobacillus spp. In others studies conducted on rats [
58] and humans [
59], proanthocyanidin-rich extracts from grape seeds showed prophylactic effects against chemotherapy-induced intestinal injury (mucositis) [
58] and significantly increased the number of
Bifidobacteria (by 0.5 log cfu/g of feces on average), while inhibiting other groups such as Enterobacteriacae (0.8 log cfu/g of feces on average) [
59].
Therefore, taking into account all the above results and previous studies, the prebiotic activity described for Goji berry extracts in this study may be a reflection of a combined dose-dependent effect of Goji berry polyphenols, which may have a stimulatory effect at certain (low) concentrations, and polysaccharides, which may either act directly as prophylactic agents, or be degraded into mono- and oligo-saccharides, which can be used as energy sources and especially building blocks for the biosynthesis of exopolysaccharides that have a protective role against the adverse gastrointestinal conditions.