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Effect of inoculation of Lactobacillus acidophilus on the level of nitrite and nitrate in yogurt containing spinach during refrigeration in 21 days

Effect of inoculation of Lactobacillus acidophilus on the level of nitrite and nitrate in yogurt containing spinach during refrigeration in 21 days. Functional Foods in Health and ABSTRACT Introduction: Yogurt is mixed with spinach in some countries. Spinach can be a probable dietary source of Nitrate and Nitrite, and further N-nitrosamine. Methods: The present study aimed to investigate the effect of inoculation of Lactobacillus acidophilus on the level of nitrite and nitrate in “yogurt containing spinach” (YS) during refrigeration yogurts. This study showed that Lactobacillus acidophilus bacteria cannot affect the amount of nitrate and nitrite in yogurt or YS at 4°C. Conclusion: Increasing the nitrite content of samples may enhance the possibility of nitrosamine formation. It is suggested that those who are at risk for all malformations caused by nitrites should not keep YS for a long time.


INTRODUCTION
The compounds nitrates and nitrites are present naturally in the environment and can be consumed by humans through meats, vegetables and drinking water.
Nitrate can convert into nitrite by bacterial activity [1].
Because of their carcinogenic effect [1][2], they may lead to some diseases including methemoglobinemia in infants [3][4] and congenital malformations in children [5], they have been considered as unwanted compounds in the diet. Furthermore, an increase in the nitrate level leads to block iodide uptake of the sodium iodide symporter [6]; also, it can lead to nutrient deficiency and raise the risk of kidney stones [7]. Many studies have indicated that nitrites can react with secondary amines and form carcinogenic compounds named Nnitrosamines in the stomach [1][2][8][9]. However, some studies have revealed that nitrite accumulation in the tissues and blood is a biological source of nitric oxide (NO) which can enhance gastroprotection and reduce blood pressure. It possesses good effects on cardiac function and participate in mitochondrial respiration [10][11]. Nitrates are found in large quantities in vegetables, so that 70-90% of dietary nitrate intake comes from vegetables [12][13][14][15][16]. Green leafy vegetables contain higher levels of nitrate and spinach is one of them [14 -15, 17].
One of the forms of spinach consumption in Iran is a combination of cooked spinach with yogurt, and it is not clear what effect has the combination of spinach with yogurt on the amount of nitrite and nitrate; furthermore, the prepared mix may be refrigerated for several days.
However, it should be noted that nitrate and nitrite are compounds that have an Acceptable Daily Intake (ADI).
The ADI for nitrate is 3.7 mg kg-1 day-1 [3,15,18] and for nitrite it is 0.07 mg kg-1 day-1 [19]. It means that the nitrate intake for a 60 kg adult should not be higher than 222 mg nitrate per day [3,[14][15][18][19]. Therefore, nitrate and nitrite intake should be minimized, especially for those who consume lots of vegetables in their diet [18].
Today, a part of yogurt production in the world is in the form of probiotics. Probiotics are live microbial food supplements that have benefits for health [20]. Some beneficial effects of probiotic consumption include improvement of digestive system health, reducing the risk of certain cancers, contributing to relief from lactose intolerance symptoms [21][22], enhancement of the immune system, synthesis and enhancement of the bioavailability of nutrients, decreasing prevalence of allergies in susceptible individuals [22], anti-diarrhea effects [21,23] and reducing serum cholesterol [21,[23][24]. In addition, the consumption of probiotic yogurt can also reduce the inflammatory, infectious, and metabolic effects of pregnancy [25]. Among the probiotic bacteria, lactic acid bacteria have been identified as the most important one [26].
Studies have shown that lactic acid bacteria are involved in the depletion of nitrate in many foods [4].
Recent findings indicate that lactobacillus can use nitrate and nitrite and produce NO in the digestive tract, which plays a role in physiologic events as told [27][28].
Yogurt is mixed with vegetables such as spinach in some countries, and there is not any study on the effect of yogurt starter and probiotic bacteria activity on the amount of nitrite and nitrate in these yogurts during the storage period in the refrigerator. Also, each kilogram of fresh spinach can contain up to 5 grams of nitrate [5,29]  and in the two groups of Y and PY this increase was seen only in the first week, and after that, the amount of nitrate was reduced. However, none of these changes was significant. Differences between values in columns signed with the same letters are non-significant Table 2 [33].

Preparation of 4 types of yogurts: To prepare
Therefore, the nitrate concentration of yogurt in this study was similar to the results of some studies [31] although was high compared to the other two studies [32,33] al. [32] and 0.002 mg kg -1 by Luf et al. [33]. A higher amount of nitrate in dairy products can be due to the water or gas used in the processing or because of the use of nitric acid as a cleaning agent in the factories [33,34].
Also, milk contamination with nitrate can occur during or after secretion. Since the nitrate of forage is carried to milk in a low amount, most nitrate contamination has a post-secretory route [33]. As Kammerer et al. showed, contamination of the drinking water of cows with nitrates could not significantly change the nitrate levels of their milk [35]. However, Baranová et al. found that after feeding potassium nitrate (KNO3) to dairy cows, a considerable increase in nitrate of milk was observed, depending on the amount of KNO3 [36]. According to Bouchard's study, increased concentration of nitrate and nitrite in milk can also be due to endotoxin-induced mastitis [37]. Therefore, although the amount of nitrate and nitrite in milk and dairy products is very low in normal conditions, for other reasons, this can be higher than usual. To find the main reason for the higher nitrate and nitrite levels in the yogurts tested in this study, further evaluation should be done.
In this study, the mean nitrate and nitrite levels  [40]. Another probable cause is the reduction of the survival of probiotics in yogurt [41] affected by factors such as pH reduction and organic acid accumulation during growth in the storage period [42]; there are some studies that show that the pH of samples of probiotic yogurt decreased during storage at 4°C, and their acidity increased [43][44][45]. Another factor can be the inhibitory effect of nitrate and nitrite on bacterial growth; it has been shown in a study that nitrite can inhibit the growth of bacteria in certain concentrations and pH [46]. yogurt [47].
The mean nitrate concentrations of two groups of Y and YS, and two groups of PY and PYS were significantly different on all measuring days; this suggests that the addition of spinach influences the nitrite levels of yogurt, which seems to be natural due to the high amount of nitrate in spinach [14]. However, the mean nitrite concentrations were almost the same in all groups and did not have a significant difference. Since the amount of spinach added to yogurts in this study was 15 percent, and according to the amount of nitrite in the cooked spinach [14], this result is explainable.