1. Introduction
The
Perilla frutescens var. nga-keemon plant (known by its Japanese name “shiso”) is cultivated in the upper northern provinces of Thailand, specifically Chiang Mai, Nan, and Maehongsorn Provinces. It is an edible fruit that is commonly used as an additive in rice, cookies, and biscuits.
P. frutescens oil (PFO) is abundant with omega-3 polyunsaturated fatty acids (ω3-PUFA), particularly α-linolenic acid (ALA) at 54–65% (
w/
w) of the total fatty acid content [
1,
2,
3]. In the human body, ALA is metabolized into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and eicosanoids, all of which are known to exert anti-inflammatory, anti-thrombotic, and various neuroprotective effects [
4,
5,
6,
7]. With regard to its anti-atherogenic properties, perilla oil has been observed to lower plasma lipids and decrease the size of fatty-streak lesions in apolipoprotein-E knockout mice [
8]. Accordingly, these properties can improve various cardiovascular and immune functions [
9,
10,
11]. Importantly, PFO is also comprised of lipophilic antioxidants, such as vitamin E, tocopherols, and tocotrienols [
3,
12,
13]. Recently, we have determined that PFO possesses analgesic, anti-inflammatory, and anti-ulcerative properties [
14], all of which can improve red blood cell (RBC) indices and protect against carbon tetrachloride-induced hepatotoxicity in animal models [
3]. Moreover, the ethanolic extract of
P. frutescens fruits is abundant with phenolics and flavonoids that have been observed to exhibit anti-inflammatory activities in tumor necrosis factor-alpha (TNF-α)-induced endothelial (EA. hy926) cells. Furthermore, this extract can also reduce oxidative stress and lipid peroxidation in human hepatocellular carcinoma (HuH7) cells [
15,
16].
At present, the food industry is focusing its efforts on developing new, healthy, and functional plant-based beverages. In fact, soybean milk (SM) possesses the highest protein content (35–50%,
w/
w) when compared with other kinds of milk [
17]. Nutritionally, SM contains fatty acids, vitamins, minerals, antioxidants, and isoflavones [
18,
19]. Accordingly, it is a suitable option for people who are allergic to cow’s milk (lactose intolerance). Importantly, the isoflavones present in plant-based beverages have been found to effectively prevent various postmenopausal syndromes, prostate cancer, and osteoporosis [
20,
21]. Additionally, the regular intake of SM protein can lower serum triglyceride (TG) and low-density lipoprotein-cholesterol (LDL-C) levels [
22]. Hence, there is an important need for the development of new functional dairy alternatives. This would include ω3-PUFA rich soybean milk. Sesame oil is an edible oil pressed from
Semen sesame and
Sesamum indicum seeds. It is known to be substantially abundant with tocopherols (particularly γ-tocopherol) and phenolic compounds. The oil exhibits interesting nutritional and biological properties [
23]. In our research, we have fortified it with SM and preserved its shelf-life using high temperature treatment (UHT) in order to meet the needs of consumers [
24]. Pasteurization is a method of food processing aimed to eliminate pathogenic bacteria, preserve most temperature-sensitive compounds (such as proteins and bioactive ingredients), and regulate the activity of mother’s milk [
25,
26,
27]. Accordingly, sensory properties and consumer acceptance scores of product prototypes are required to optimize sensory quality based on the nine-point verbal hedonic box scale [
28,
29,
30]. The fortification of PFO in SM has made it a multi-purpose drink that combines bioactive and nutraceutical properties. Therefore, it has received a significant amount of attention from consumers. We conducted our research in order to develop a pasteurized PFO-SM drink, identify its sensory characteristics and level of consumer acceptance, and to clinically investigate the relevant health outcomes among healthy participants after consumption of our PFO-SM product.
4. Discussion
Sesame seed oil is used primarily in salad oil, and has served as both a frying medium and a nutrient additive in beverages. In evaluating the quality of certain edible vegetable oils, the nutritional indexes of sesamin oil were reported with regard to total saturated fatty acids (SFAs), atherogenic fatty acids, including SFAs with 12, 14, and 16 carbon chains, monounsaturated fatty acids, and PUFAs such as LA and ALA [
45]. Sesame oil is derived from the benne plant (
Sesamum indicum) and is commonly consumed in African-American cuisine. This is the first study to focus on developing a functional pasteurized perilla fruit oil-fortified soybean milk and identify the relevant sensory characteristics, along with its level of consumer acceptance. Herein, we have developed a pasteurized PFO-fortified SM drink product that is rich in ALA and then identified its degree of sensory acceptance by consumers. According to the resulting evaluations, most of the consumers in the test expressed an interest in buying the product (68%). These consumers stated that they were willing to pay 0.45–0.60 US dollars (15–20 baht) for the 1.0% PFO-SM product. Assessments were made according to the price, taste, brand, and health benefits of the product. We then determined the outcomes of the consumer evaluations with regard to the product’s color, odor, flavor, sweet taste, additive taste, and texture. In comparison, the price of commercially available and high temperature manufactured black sesame-fortified soybean milk (180 mL/carton) is 0.28–0.30 US dollars (9.50–10 baht), which is 1.5 = 2 times cheaper than pasteurized PFO-SM. In the future, the production of PFO-SM beverages, on both commercial and industrial scales, will pull down the manufacturing costs and increase the product’s cost effectiveness as it relates to its potential benefits to human health. With regard to the nutritional facts, our pasteurized PFO-SM contains less calories, fats, and salts but does contain higher temperature-sensitive bioactive compounds than the BS-SM product. This is indicative of greater nutraceutical content and improved health benefits. Notably, the high temperature treatments (120–180 °C) associated with the manufacturing of sesame oil products via roasting, frying, and possibly sterilization can influence the PUFA ratios of C16:0, C18:0, C18:1, and C18:2 chains and significantly decrease the mineral content [
46]. Dhibi and colleagues reported that fried sesame oil had higher amounts of atherogenic lipid products (such as trans fats and conjugated linoleic acid) than fresh sesame oil [
47]. More importantly, a previous study has supported the determination that the pasteurization (62.5 °C for 30 min) of human milk did not affect total fat content and the percentage of compositions of saturated fatty acids, monounsaturated fatty acids, and ω3- and ω6-PUFA levels. However, sterilization (120 °C for 30 min) was found to significantly reduce the amounts of available fatty acids, such as LA and arachidonic acid (AA) [
48,
49]. Nevertheless, both pasteurization and sterilization methods induced oxidative degradation and increased amounts of propanal (or MDA) of 1% linseed oil-enriched dairy products through the chain-reaction peroxidation of the PUFA existing in linseed oil [
50].
Though knowledge of consumer perception to food and drink products is growing rapidly at present, relevant knowledge and health outcomes established through the consumption of PFO-SM drinks are now being investigated using randomized clinical trials. Taken together, we chose to add 1% (
v/
v) PFO, rather than 0.5% (
v/
v) PFO, to SM, so that we would obtain a more concentrated PFO-SM drink (higher ALA abundance), greater flavor and taste (as described above), and improved biological and pharmacological effects [
3]. In the present study, 1% PFO-SM drink was chosen to be tested among healthy subjects and the findings were compared with those of a basic SM product and a commercial BS-SM product. Likewise, the DI group was included in this study to confirm that the different outcomes possibly resulted from the consumption of either soybean milk or perilla oil, or a combination of the two. In our findings, no significant results were observed when comparisons were made between the DI group and the SM group. This suggested that the SM compositions did not influence, or change, the relevant health outcomes and levels of the blood biomarkers in the participants who consumed the drinks for 30 days. Indeed, we found that consumption of PFO-SM for 30 days lowered levels of serum triglycerides and red cell ROS, while increasing serum antioxidant activity in healthy subjects when compared to basic SM and a commercial BS-SM product. We have previously reported an abundance of unsaturated fatty acids, including ω3-ALA, ω6-linoleic acid, and ω9-oleic acid (57, 20 and 13% of total fatty acids, respectively); α- and β-tocopherols (2.5 and 4.95 mg%, respectively), γ-tocotrienol (4.37 mg%), and δ-tocopherol (1.28 mg%), in PFO. This would indicate its ability to exert protective effects on carbon tetrachloride-induced hepatotoxicity in rats [
3]. In addition, PFO also exhibited analgesic and anti-inflammatory effects in rats [
14]. In the human body, ALA is metabolized to PUFAs, such as EPAs and DHAs, while LA is a precursor for the biosynthesis of arachidonic acid (AA), via the process of catalysis by elongase and reductase [
51,
52]. Subsequently, both EPA and AA are further converted to the three main classes of eicosanoids, including prostaglandins, thromboxanes, and leukotrienes, which are known to exhibit biological and pharmacological functions in the human body [
53]. Though ALA and LA are competitively metabolized using the same enzymes, eicosanoids obtained from ALA and LA act in opposing ways in the human body [
54]. Accordingly, EPA-derived eicosanoids perform anti-inflammatory and anti-thrombotic activities, while AA-derived eicosanoids reveal pro-inflammatory and pro-thrombotic activities and vice versa [
10,
55]. Several studies have demonstrated that the intake of perilla oil increased the plasma levels of EPA and DHA and was associated with lower incidences of cardiovascular disease, while overconsumption of ω6-PUFA was highly associated with the prevalence of chronic inflammatory diseases and abnormal clotting [
1,
2]. Therefore, maintaining a balanced ratio of ω6:ω3 at 4–5:1 in one’s diet is important in preventing incidences of chronic inflammatory diseases [
10,
56]. Certainly, PFO would be recommended as a good resource for supplying ω3-PUFA within a well-balanced ratio of ω6:ω3 PUFA.
Notably, there were more female subjects than male subjects (ratios of 2:1-4:1) in all four groups participating in this study. Nevertheless, no significant differences of the participants’ ages among the four groups and between the groups were observed. In the present study, the subjects were in good physical health and their weights were considered appropriate for their age and sex. Hematological and serum biochemical profiles were normal and there were no major differences or significant differences between the two groups in terms of sex distribution at the baseline and after consumption of the drinks. Consequently, any potential confounding factor pertaining to age or gender would not have influenced the outcomes of our study. Some previous studies have reported that sesame oil, rich in PUFAs, such as LA, inhibited the growth of HT-29-malignant human colon cancer cell lines and thymoma cells in C57BL/6 mice before and after being challenged with EL4 lymphoma cells [
57,
58]. Interestingly, after consumption of a sesamin oil-supplemented diet, LA is metabolized to ω6 dihomo-γ-linolenic acid product exhibiting antioxidant and anti-inflammatory activities and subsequent hepatoprotective effects against hepatic ischemia-reperfusion injury [
59]. Surprisingly, sesame oil mixed with (10%,
v/
v) ALA-rich
Garden cress oil revealed a significant decrease in serum levels of TC, TG, and LDL-C in rats [
60]. Contrastingly, we found that the consumption of black sesame oil-fortified soybean oil (BS-SM) increased serum levels of atherogenic lipids, such as TC, TG, and LDL-C, in healthy human subjects. According to the lipid profiles, PFO-SM decreased the serum levels of TG significantly; while SM and BS-SM effectively increased serum TG levels. Though there were significant alterations in some biochemical parameter levels in the PFO-SM group, when compared with the BS-SM group, the changes were not deemed to be clinically relevant. In spite of small fluctuations, the values remained within normal clinical ranges. Soung and colleagues have revealed that post-menopause women consuming 60 mg isoflavone-supplement soy protein (5 g) drinks for 1 y showed an increase in RDW (a marker of reticulocytes) [
61]. Interestingly, mild-asthmatic patients (11F, 4M; mean age, 61.0 y) who were fed a PFO salad (14.65 ± 1.41 g/d) for 4 w showed significant decreases in serum total cholesterol, LDL-C (
p < 0.05), and triglyceride levels suggesting lipid-modulating effects [
62]. Consistently, type 2 diabetic patients (20M, 30F, age 63.84 ± 9.67 y) who were orally administered PFO capsules for 6 months showed decreases in serum TC, LDL-C (
p < 0.05), and TG levels [
63]. It has been postulated that the ω3-ALA present in PFO could be a key ingredient in effectively decreasing the levels of blood lipids, particularly TG. This would possibly have occurred by the activation of fatty acid oxidation or/and competitive inhibition of intestinal lipid absorption. Hypothetically, the intake of ALA would activate plasma membrane fatty acid translocase (FAT/CD36) to translocate fatty acyl CoA across the membrane [
64,
65]. Moreover, consumption of essential ALA was found to increase maximal fat oxidation in athletes [
66]. Several studies found that ω3-PUFA increased the expression of carnitine palmitoyl transferase I, which is known as the rate-limiting enzyme involved in fatty acid oxidation. However, ω3-PUFA also suppressed the expression of acetyl-CoA carboxylase known as the rate-limiting enzyme in fatty acid synthesis [
67,
68]. Furthermore, administration of cold-pressed perilla oil was observed to significantly reduce the expression of the peroxisome proliferator-activated receptor (PPAR)γ and fatty acid synthase (FAS) activities in incidences of high-fat diet-induced obesity in mice, leading to an inhibition of hepatic steatosis [
69]. Consumption of PFO-SM by healthy volunteers for 30 days did not produce any changes in levels of hematological parameters or biochemical parameters, except for a significant decrease in serum AST. Consistently, our previous study has reported that oral administration of PFO for 90 d significantly decreased serum levels of ALT, ALP, and particularly AST in carbon tetrachloride-induced hepatotoxicity in rats, which would be indicative of its hepatoprotective effect [
3].
Thies and colleagues demonstrated that healthy subjects in group A (4M, 4F), group B (5M, 3F), and group C (3M, 4F), who ingested the ALA blend (a mixture of palm, sunflower seeds, and flaxseed oil), DHA, and fish oil (a mixture of EPA and DHA) for 12 weeks were not associated with changes in total and differential WBC numbers or phagocytotic activity [
70]. Our findings have revealed that PFO-SM consumption significantly enhanced phagocytosis when compared with SM and BS-SM, which could be attributed to bioactive phenolic compounds, but not 3ωPUFA
per se [
71].
With regard to oxidative stress and antioxidant capacity profiles, similarly, there were no significant differences between the two groups in terms of sex distribution at the baseline and after consumption of the drinks. Nevertheless, the two groups differed with regard to the efficacy of the PFO-SM drinks, although blood analysis excluded any possible confounding effect of sex differences in the outcomes of these parameters. Indeed, we are confident that PFO-SM exerted ROS-scavenging properties in RBC and plasma compartments, possibly by directly using conjugative double bonds to absorb the persisting ROS and/or to enhance antioxidant defense system in the body. Marinkovic and colleagues have demonstrated that the pasteurization process did not influence total antioxidative properties, but did change specific components and decreased the SOD and glutathione peroxidase activities of raw human milk per se [
26]. In our study, we have observed a significant decrease in red cell ROS along with an increase in the serum antioxidant capacity among members of the PFO-SM group. We have demonstrated that PFO is abundant with lipid-soluble antioxidants, including tocopherols and tocotrienols [
3]. Evidently, cold-pressed PFO was found to lower ROS levels in ultraviolet-induced normal human dermal fibroblasts [
72]. Moreover, Lee and colleagues have illustrated that PFO exhibited a greater degree of DPPH radical-scavenging activity than oleic acid-rich olive oil and linoleic acid-rich corn oil [
73]. Similarly, the administration of α-tocopherol for 3 months significantly reduced levels of red cell ROS and serum lipid-peroxidation products in β-thalassemia intermedia patients [
74]. From a current randomized, double-blind clinical study, healthy elders who consumed PFO (0.88 g ALA rich)-fortified ponkan powder (2.91 mg nobiletin abundant) for 12 months revealed significantly higher cognitive index scores than the PFO group. This would suggest that a pro-cognitive effect is accompanied by increases in ALA and DHA levels in red cell membranes, serum brain-derived neurotropic factor levels, and biological antioxidant capacity [
75]. Likewise, the supplementation of α- and γ-tocopherols for 6 weeks significantly decreased levels of plasma lipid peroxides and MDA in subjects diagnosed with metabolic syndrome [
76]. Moreover, cold pressed-PFO treatment effectively suppressed oxidative stress and decreased SOD activity in PC12 cell cultures [
77]. Surprisingly, the intake of dietary PFO increased serum antioxidant capacity and ALA levels in the RBC plasma membrane of healthy volunteers who were within an age range of 64-84 years old [
78]. Taken together, the findings suggest that PFO-SM abundant with fat-soluble antioxidants, including tocopherols, tocotrienols, and PUFAs, could efficiently detoxify the ROS existing in blood components and prevent oxidative stress-related disorders.
Herein, we are attempting to introduce perilla fruit oil into the food and drink industry as a potential way to substitute PUFAs for the saturated fatty acids found in soybean milk. Importantly, it represents a cheaper plant-derived form of ω3-PUFA than deep-sea water fish oil. We would like to specifically highlight its potential health promoting benefits. In terms of limitations, PFO is hardly soluble in an aqueous SM base, so it would be very difficult to increase the amounts of ALA and other active ingredients as a way of achieving a greater degree of efficiency. Most of the subjects enrolled in this study were women who may have been affected by their menstrual cycles, resulting in blood loss and possible changes in red cell indices. Lastly, the study time was only one month, which may have been too short to observe considerable differences in these parameters.