The aim of this study was to evaluate the effect of a 2% chia seed oil addition to natural yogurt on its quality and to determine whether chia seed oil can be used as an additive in fermented milk products. The dominant species of microorganisms found in yogurt was
Recent years have seen an increased interest in fermented milk drinks, particular in yogurt-based formulations. More and more consumers are reaching for it in their daily diet, and their annual intake in the European Union ranges from 6.5-19 kg per person, with the largest share of yogurt containing fruit (Agriculture and Agri-Food Canada –Report
Yogurt is defined as a product resulting from milk by fermentation with a mixed starter culture consisting of
Chia (
Chia seeds are also distinguished by a high content of vitamins E and minerals compared to other seeds. They contain a relatively high content of vitamin E, about 8 mg per 100g (Silva da
The most abundant polyphenols in chia seeds are phenolic acids, including rosmarinic, caffeic and ferulic acid (Rahman
In 2009, the European Food Safety Authority published a positive decision about introducing chia seeds on the market. The decision permits adding 5% chia seed to bread products. Four years later the Commission Implementing Decisions recommended an extension of the use of chia seeds to no more than 10% in baked products, breakfast cereals, fruit, nut and seed mixes, and the marketing of pre-packed chia seeds. Moreover, food producers are obliged to inform consumers that the recommended daily intake of chia seeds is no more than 15 g per day. Still, chia seed addition to fermented milk products like yogurt and fermented milk products flavored with fruit components, fruit spread or cereal and/or pulse-based ready-to-serve (RTS) meals, and fruit preparations has not yet been approved. The application of 3% chia seed in chocolate bars is currently pending by the EFSA [EU website].
The aim of this study was to investigate how the addition of 2% chia seed oil would affect the quality of natural yogurt. The article presents the changes in fatty acid and sterol compositions in the fat fraction of the analyzed yogurt and describes how the addition of chia seed oil to natural yogurt does not change the pH or LAB population over a 4-week storage period, or the sensory quality of natural yogurt. This article focuses on the addition of chia oil to natural yogurt in contrast to other articles, which mainly describe the addition of chia seeds to yogurt and their impact on product quality.
Chia seeds were produced by the company My-Vita and purchased at the organic food store in Warsaw (Poland) in 2016. The oil extraction from chia seeds was carried out according to the recommendations of the manufacturer of the PITEBA oil press. The extraction efficiency of PITEBA oil expeller was 80%, oil yield was 0.9 l·h-1. Initially, the seeds were watered down with 50 g of water per kilogram. Water was added in small portions until the seeds became sticky, uniform, and formed a mucilage mass. The seeds were stored in a closed container at room temperature for 24 h. After this time the seeds were transferred to the hand press. Then the wick was burned from the paraffin container to gently warm the beans inside the press. After 10 min the process started by turning the crank clockwise (the pressure exerted to extract the oil was unknown). The oil flowed through the outlet opening in the front bottom of the tube. The oil was collected in a 500 mL glass beaker and after cooling it was centrifuged at 10000 rpm for 10 min. Subsequently, the clear (top) oil was carefully collected over a nitrogen gas atmosphere, sealed and stored at a cooling temperature (4 °C).
850 g of UHT milk with a 1.5% fat content of OSM Wart-Milk (Poland) were weighed into 6-sterilized 1-liter Schott bottles and then 17 g of skimmed powdered milk from S.M. Gostyń(Poland) were added. The whole mass was intensively mixed using a laboratory mechanical homogenizer (Silverson L4R, England) until the milk granules dissolved completely (4000 rpm for 15 min at 25 ºC). 2 g of chia seed oil were added to the batches of enriched yogurt to partially replace milk fat. The level of chia seed oil addition was earlier investigated and did not lead to de-lamination of the product. The whole mass was mixed once again (4000 rpm for 10 min at 25 ºC) and incubated in a 50 °C water bath for 30 min, stirring occasionally.
After this time, 0.002% of yogurt starter cultures was added to all the bottles (YC-180 obtained from Chr. Hansen, Poland), followed by intensive stirring for 5 min. The prepared product was then poured into 40 mL jars (5 jars from each bottle) and into the thermostat (temperature 45 ºC) to form a curd yogurt. Incubation was carried out for 4 h until the yogurt curd was suitably formed, uniform and dense. The yogurt was then cooled and stored at refrigerated temperatures for 0, 14, and 28 days.
In order to extract the fat fraction required for the determination of fatty acids and sterols in yogurt, the samples were weighed into Falcon centrifuge tubes respectively:
For determination of fatty acids: 1.5 g of enriched yogurt and 3 g of natural yogurt.
For determination of sterols: 6 g of enriched yogurt and 12 g of natural yogurt.
Afterwards the mixture of chloroform: methanol (2:1, v:v) was added to the enriched yogurt (12 mL) and to natural yogurt (20 mL). The samples were centrifuged at 9000 rpm for 10 min. The bottom chloroform layer was collected and filtered into the vials. The samples were evaporated under a nitrogen stream and weighed after complete drying.
The extracted fat samples were weighed (about 50 mg) and dissolved in 2 mL of hexane. Next, 0.5mL of 2M KOH in methanol was added to the samples, afterwards the samples were shaken and left to for 1 h. After the process of transesterification 1 mL of the upper layer was collected carefully and transferred to a glass vial. The sample was evaporated in a stream of nitrogen and 0.5 mL hexane was added. Each fat sample was analyzed in triplicate. The separation of the fatty acid methyl esters was performed with the use of a gas chromatograph coupled with mass spectrometry Shimadzu – QP-2010S and capillary column ZB FFAP Phenomenex (30 m x 0.25 mm x 0.25 μm). Column temperature procedure: initial 60 °C for 3min, temperature rise at 4 °C·min-1to 230 °C and held for 5 min. Injector and ion source temperature were as follows: 230 °C and 240 °C. The carrier gas was helium, with a flow of 1.14 mL·min-1. The interface temperature of GC-MS was 225 °C. The ionization energy was 70 eV. The Total Ion Monitoring (TIC) was used to detect fatty methyl esters (m/z ranged 50–500). The qualitative analysis of the fatty acid methyl esters was made on the basis of a comparison of their retention times with those of available standards and mass spectra as well as literature data. The results were presented as percentage of each fatty acid in the total profile of fatty acids. Each sample was analyzed in triplicate.
The extracted fat samples were weighed (about 150 mg) and dissolved in 2 mL of hexane. Than 100 µL of 5α-Cholestan-3β-ol standard (concentration 11.93mg in 25 mL of chloroform) were added to the samples. Next 0.5 mL of 2M KOH in methanol was added to the samples; afterwards the samples were shaken and left for 1 h. After the process of transesterification 1 mL of the upper layer was collected carefully and transferred to a glass vial. The sample was evaporated in a stream of nitrogen and 100 µL of silylating reagent (BSTFA + TMCS, 99:1) and 100 µL pyridine were added. The prepared sample was shaken and left for 24 h at room temperature according to the derivatization process. Next 0.5 mL of hexane was added and the trimethylether sterol content was analyzed. The separation of sterol derivatives was performed with the use of GC coupled with a Shimadzu – QP-2010S and capillary column ZB 5MS Phenomenex (30 m x 0.25 mm x 0.25 μm). Column temperature procedure: initial 60 °C for 3 min, temperature rate 15 °C·min-1to 250 °C, second temperature rate 3 °C·min-1to 310 °C and held for 10 min. The injector and ion source temperature were 250 °C and 240 °C, respectively. Thecarrier gas was helium at a flow of 0.47 mL·min-1. The interface temperature of the GC-MS was 250 °C. The ionization energy was 70 eV. The Total Ion Monitoring (TIC) was used to detect sterols (m/z ranged 100–600). The qualitative analysis of trimethylesters was made on the basis of a comparison of their retention times with the retention time of available standards and mass spectra as well as literature data. The internal standard 5α-Cholestan-3β-ol was used to quantify sterols and the results were presented in mg·100g-1of fat. Each sample was analyzed in triplicate.
Preparations of samples for microbiological analysis, as well as dilutions, were performed according to ISO 6887–5:2010. MRS agar (Merck, Poland) was used to quantify
The pH measurements in the yogurt samples after fermentation and storage (7, 14, 21, and 28 days) ware determined with the use of a pH meter HI 931400 Microprocessor pH-metr (HANNA Instruments company). All experiments were repeated three times.
The yogurt samples were subjected to a sensory evaluation by 15 panelists. The samples were evaluated after 24 h of fermentation and storage at 4 ºC for 14 and 28 days. Each panelist was trained and asked to rank all samples for color, surface gloss, flavor, fatty flavor, taste, fatty taste, uniformity of structure, texture, foreign tastes, acidity and acceptance using a 5-point hedonic scale, ranging from 1 (spoiled food sample) to 5 (the best feature, meeting the sensory requirements). The yogurt samples were coded, and samples were given to the panelists in an anonymous and random manner.
The results were analyzed statistically using the Excel and Statistica 12 programme, where a one way Anova variance analysis was performed (Tukey’s post-hoc score) (student grouped medium).
Chia seed oil contained five different fatty acids (
The profile of fatty acids in chia seed oil (n=3, mean ± SD)
Fatty acids | Profile (%) | Results reported by other authors (%) | |
---|---|---|---|
9.69 ± 0.12 | |||
Palmitic acid | C16:0 | 6.52 ± 0.08 | 7.47% (Segura-Campos |
Stearic acid | C18:0 | 3.17 ± 0.05 | 0.3% (Segura-Campos |
90.3 ± 0.12 | 90.3% (Coelho and Salas-Mellado, |
||
6.66 ± 0.08 | |||
Oleic acid | C18:1 |
6.66 ± 0.08 | 7.2% (Ayerza and Coates, |
83.6 ± 0.19 | 79.5% (Coelho and Salas-Mellado, |
||
Linoleic acid | C18:2, |
17.10 ± 0.09 | (18.8-20.4%) (Rossi |
α-linolenic acid | C18:3, |
66.54 ± 0.28 | (62.0-68.5%) (Rossi |
The results published by Ayerza and Coates (
The same amount (90.3%) of unsaturated fatty acids in chia seed oil was detected by Coelho and Salas-Mellado (
Rossi
Ayeza and Coates (
The most abundant fatty acid in the profile of yogurt was palmitic acid. In the group of saturated fatty acids stearic and lauric acid were present in high amounts (about 11.6% and 10.42%, respectively) (
The profile of fatty acids in natural yogurt (n=3, mean ± SD)
Fatty acid | Profile [%] | |||
---|---|---|---|---|
Storage day | ||||
0 | 14 | 28 | ||
65.59 ± 0.17 |
67.54 ± 2.46 |
66.18 ± 1.78 |
||
Butyric acid | C4:0 | 0.47 ± 0.17 |
0.49 ± 0.21 |
0.45 ± 0.28 |
Caprylic acid | C6:0 | 1.76 ± 0.09 |
1.94 ± 0.22 |
1.74 ± 0.21 |
Capric acid | C8:0 | 2.99 ± 0.54 |
3.18 ± 0.72 |
2.94 ± 0.26 |
Lauric acid | C10:0 | 10.42 ± 1.94 |
11.65 ± 0.99 |
11.30 ± 0.37 |
Myristic | C12:0 | 0.99 ± 0.04 |
0.98 ± 0.04 |
0.97 ± 0.02 |
Palimc acid | C16:0 | 37.26 ± 0.90 |
37.85 ± 1.07 |
37.53 ± 0.78 |
Margaric acid | C17:0 | 0.55 ± 0.03 |
0.51 ± 0.05 |
0.51 ± 0.06 |
Stearic acid | C18:0 | 11.62 ± 1.17 |
11.06 ± 0.19 |
11.37 ± 0.19 |
31.45 ± 1.40 |
30.25 ± 1.38 |
30.41 ± 0.78 |
||
|
|
|
||
Pentadecanoic acid | C15:1, |
1.22 ± 0.03 |
1.22 ± 0.12 |
1.17 ± 0.06 |
Palmitoleic acid | C16:1, |
1.84 ± 0.52 |
1.89 ± 0.08 |
1.96 ± 0.21 |
Oleic acid | C18:1, |
25.27 ± 1.06 |
24.68 ± 1.43 |
25.52 ± 0.97 |
Linoleic acid | C18:2 |
2.34 ± 1.13 |
1.95 ± 0.49 |
1.83 ± 0.16 |
α-linolenic acid | C18:3 |
1.08 ± 0.63 |
0.59 ± 0.24 |
0.76 ± 0.41 |
a–c Different superscripts within a row show significant differences during storage (P < 0.05) (according to one way ANOVA and Tukey test)
The saturated fatty acid composition was dominant in the fatty acid profile of yogurt (65.60% on the day of manufacture, 67.54% after 14 days of storage and 66.18% after 28 days of storage). Unsaturated acids accounted for about 30% of the total profile, of which only 2.5-3.3% were polyunsaturated acids (linoleic acid and α-linolenic) (
Paszczyk
The highest amount in the fat fraction of yogurt with 2% chia seed oil was α-linolenic acid (45.33%) and it was the predominant component of the polyunsaturated fatty acid group. In the PUFA group the content of linoleic fatty acids was very high – 12.93%. Among the monounsaturated fatty acids the most abundant was oleic acid with the content of 12.41%. In the group of saturated fatty acids, palmitic acid was predominant (16.53%), followed by stearic acid (5.79%) and lauric acid (3.46%) (
The profile of fatty acids in natural yogurt with addition of 2% chia seed oil (n=3, mean ± SD)
Fatty acid | Profile [%] | |||
---|---|---|---|---|
Storage day | ||||
0 | 14 | 28 | ||
28.16 ± 2.24 |
34.53 ± 2.55 |
34.73 ± 2.82 |
||
Butyric acid | C4:0 | 0.19 ± 0.05 |
0.23 ± 0.13 |
0.20 ± 0.11 |
Caprylic acid | C6:0 | 0.55 ± 0.14 |
0.67 ± 0.15 |
0.70 ± 0.15 |
Capric acid | C8:0 | 0.81 ± 0.16 |
1.21 ± 0.21 |
1.09 ± 0.15 |
Lauric acid | C10:0 | 3.46 ± 0.43 |
4.72 ± 0.42 |
4.72 ± 0.50 |
Myristic | C12:0 | 0.28 ± 0.03 |
0.37 ± 0.11 |
0.39 ± 0.03 |
Palimc acid | C16:0 | 16.53 ± 1.18 |
20.06 ± 1.21 |
19.85 ± 1.45 |
Margaric acid | C17:0 | 0.27 ± 0.02 |
0.33 ± 0.03 |
0.33 ± 0.03 |
Stearic acid | C18:0 | 5.79 ± 0.32 |
6.75 ± 0.47 |
6.94 ± 0.62 |
71.35 ± 2.43 |
64.28 ± 2.28 |
63.59 ± 2.63 |
||
Pentadecanoic acid | C15:1 | 0.40 ± 0.06 |
0.53 ± 0.06 |
0.52 ± 0.40 |
Palmitoleic acid | C16:1 |
0.68 ± 0.09 |
0.87 ± 0.15 |
0.91 ± 0.13 |
Oleic acid | C18:1 |
12.41 ± 0.74 |
13.66 ± 1.27 |
14.79 ± 1.78 |
Linoleic acid | C18:2 |
12.93 ± 0.44 |
11.53 ± 0.49 |
11.29 ± 0.79 |
α-linolenic acid | C18:3 12,15 |
45.33 ± 2.63 |
38.22 ± 2.06 |
36.59 ± 3.35 |
a–c Different superscripts within a row show significant differences during storage (P < 0.05) (according to one way ANOVA and Tukey test)
During 28 days of storage, the yogurt with the 2% addition of chia seed oil showed a reduced content of unsaturated fatty acids. On the first day of storage the total content of unsaturated fatty acids was 71.4%, after two weeks of storage it was significantly lowered to 64.3%, and after a moth of storage the content was only 63.6% (
Worth mentioning is the fact that ratio of ω6:ω3 fatty acids in enriched yogurt ranged from 0.28-0.31, which is within the EU recommendations of 0.25-1.00. In the average European diet the proportion of ω6:ω3 acids is on average 20:1, thus causing a high risk for cardiovascular diseases and cancers. The same observations were mentioned by Kargulewicz
Four different sterols were determined in chia seed oil and the total content of sterols was 4.56 g per 100 g of oil (
Sterol contents (mg·100g-1 of oil) in chia seed oil (n=3, mean ± SD)
Sterol | g·100g-1 of oil | Results reported by other authors g·100g-1 of oil |
---|---|---|
Campesterol | 0.54 ± 0.08 |
|
Stigmasterol | 0.18 ± 0.02 |
|
β-sitosterol | 3.64 ± 0.01 |
3.5-6.0 Alvarez-Chavez |
5∆-avenasterol | 0.20 ± 0.01 |
|
Total sterols | 4.56 ± 0.01 |
4.4-5.0 Alvarez-Chavez |
a–c Different superscripts within a column show significant differences (P < 0.05) (according to one way ANOVA and Tukey test)
In mixtures of seeds available on the market the average content of phytosterols ranged from 4.4-5.0 g per 100 g of oil. Alvarez-Chavez
In the natural yogurt only two sterols (cholesterol and 5∆-avenasterol) were identified (
Sterol contents in natural yogurt (g·100g-1 of extracted fat) (n=3, mean ± SD)
Sterol | g·100g-1 of extracted fat | ||
---|---|---|---|
Storage day | |||
0 | 14 | 28 | |
Cholesterol | 1.20 ±0.05 |
1.20 ± 0.07 |
1.06 ± 0.03 |
5∆-avenasterol | 0.06 ± 0.01 |
0.12 ± 0.03 |
0.15 ± 0.03 |
Total sterol | 1.23 ± 0.05 |
1.32 ± 0.07 |
1.21 ± 0.05 |
a–c Different superscripts within a row show significant differences during storage (p < 0.05) (according to one way ANOVA and Tukey test)
Talpur
In the fat fraction of enriched yogurt, 5 different sterols were detected (
Sterol contents in yogurt enriched in 2% chia seed oil (g·100g-1 of extracted fat) (n=3, mean ± SD)
Sterol | g·100g-1 of extracted fat | ||
---|---|---|---|
Storage day | |||
0 | 14 | 28 | |
Cholesterol | 1.26 ± 0.24 |
1.20 ± 0.20 |
1.16 ± 0.24 |
Campesterol | 0.25 ± 0.04 |
0.25 ± 0.03 |
0.17 ± 0.08 |
Stigmasterol | 0.09 ± 0.04 |
0.10 ± 0.03 |
0.07 ± 0.03 |
β-sitosterol | 1.66 ± 0.26 |
1.59 ± 0.33 |
0.96 ± 0.36 |
5∆-avenasterol | 0.08 ± 0.04 |
0.06 ± 0.02 |
0.07 ± 0.06 |
Total sterol | 3.34 ± 0.07 |
3.20 ± 0.19 |
2.84 ± 0.09 |
a–c Different superscripts within a row show significant differences during storage (P < 0.05) (according to one way ANOVA and Tukey test)
The results of the study described in this paper are difficult to discuss because there is no information on the effect of enriching yogurt with chia seed oils rich in phytosterols in the literature. It is worth noting, however, that one of the best oils for the enrichment of sterols among the existing oils on the market is chia seed oil. In comparison with the results of Kopeć
Based on the pH results it was found that the 2% addition of chia seed oil did not affect the pH of the yogurt. This parameter slightly decreased during storage, especially during the first 14 days, in which it decreased by 0.2 in natural yogurt. During the next 14 days of storage only a 0.07 decrease in pH was reported (
Acidity changes in natural and enriched yogurt during 28 days of storage (n=3, mean ± SD)
Storage days | Yogurt type | ||
---|---|---|---|
Natural | Enriched with chia seed oil | ||
pH | 0 | 4.07 ± 0.14 |
4.10 ± 0.21 |
14 | 3.87 ± 0.20 |
3.93 ± 0.04 |
|
28 | 3.80 ± 0.08 |
3.87 ± 0.01 |
a–c Different superscripts within a row show significant differences during storage (P < 0.05) (according to one way ANOVA and Tukey)
On the day of production, natural yogurt was characterized by a high average score (above 4.8) of the following characteristics: gloss, lack of taste and odor of fat, structure uniformity and sweetness (
Changes in average ratings of natural yogurt and enriched yogurt during 14 and 28 days of storage (n=3, mean ± SD) a–c Different superscripts within a columns show significant differences after storage (P < 0.05) (according to one way ANOVA and Tukey)
Changes of characteristics measured in natural yogurts after 14 and 28 days of storage
The enriched yogurt was characterized by lower sensory acceptability compared to natural yogurt. The overall evaluation of the sensory characteristics of the enriched yogurt on production day showed that the product had no sensory features that prevented its consumption, and the average score was 3.9 (
Changes in average scores of characteristics measured in enriched yogurts after 14 and 28 days of storage
Comparing the overall scores for natural and enriched yogurt (
Similar observations were made by other authors in natural yogurt (Pikul and Wójtowski,
The number of lactobacilli on the first day of storage of natural yogurt was 6.3 log CFU·g-1, and in enriched yogurt it was 6.2 log CFU·g-1. For comparison, the
Yogurt bacteria population in yogurt (log CFU·g-1) (n=3, mean ± SD)
Yogurt bacteria | Storage days | ||
---|---|---|---|
0 | 14 | 28 | |
Natural yogurt | |||
6.3 ± 0.09 |
6.1 ± 0.18 |
6.3 ± 0.11 |
|
6.3 ± 0.21 |
7.4 ± 1.14 |
9.0 ± 0.1 |
|
Enriched yogurt | |||
6.2 ± 0.08 |
6.0 ± 0.16 |
6.1 ± 0.07 |
|
6.1 ± 0.23 |
7.2 ± 1.0 |
8.8 ± 0.36 |
a–c Different superscripts within a row show significant differences during storage (P < 0.05) (according to one way ANOVA and Tukey)
The results from our study show that chia seed oil might be used as an additive to yogurt. The nutritional quality of enriched natural yogurt was shown to have been improved due to a high content of polyunsaturated fatty acids (mainly by linoleic and α-linolenic acid) and phytosterols (2 g of phytosterol in 100g of fat fraction). Moreover, during a 4-week storage period, the physical and sensory properties of enriched yogurt were not changed significantly compared to natural yogurt and the amount of total LAB was not reduced in the analyzed yogurts, which proved the high stability of the enriched products.
These results indicate that chia seed oil can be used by the food industry to enrich yogurt, considering the fact that the addition of chia seeds to fermented milk products is not permitted in EU. The use of chia seed oil can be seen as the good alternative to the chia seed addition. The results of our study may open new opportunities for the design of fermented products containing large amounts of phytosterols, which decrease cholesterol levels in human blood and lower the risk of cardiovascular diseases. Moreover, consumption of this kind of enriched yogurt may protect people from neurodegenerative diseases due to its high concentration of ω-3 fatty acids.