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Abstract The mixed fermentation is based on the raw material of raspberry and mulberry. The research is to explore the sticking point for the fermentation progress. To attain the optimized fermentation condition which gained by integrating fermentation data， the concentration of pectinase should be 80 mg/L， the mass fraction of Lactobacillus， Acetobacter and yeast should be 3%， 1% and 3%， respectively， the fermentation temperature should be 28 ℃， the initial sugar content should be 24°， and the duration of fermentation should be 16 d . After the fermentation， sugar content should be adjusted to 25°， and the products should be stored for 3 d in the condition of 15 ℃. The Raspberry-mulberry leaven containing 8.5×108 probiotics per milliliter and 61.2 g total acid per liter should be diluted with various proportions of water to drink according to different habits of customers[1]. The volume ratio of leaven and water should be at least 1∶1 and at most 1∶3.
Key words Fermentation； Mixed； Optimization
Mature mulberry contains following substances per 100 g： heat 57 kcal， dietary fiber 4.1 g， protein 1.7 g， calcium 37 mg， carbohydrate 13.8 g， iron 0.4 mg， sodium 2 mg and potassium 32 mg， but no cholesterol and saturated fat. Mulberry， considered to be a nutritious fruit for liver and kidney in Chinese medicine， is often utilized as diuretic health care food what has been recorded in Compendium of Materia Medica， a medical book in ancient China[2].
Raspberry， one of the third generation fruit popular in the world at present， has a high level of nutrition， pigment and Vitamin C and 21 unique ingredients in it[3]. Raspberry could relieve the pain due to drunk， sore throat， multiple abscesses and mastitis.
Materials and Methods
Materials
Raspberry， sold in local market； mulberry， sold in local market； pectinase， purchased from Hunan Yotel Biochemical Co.， Ltd.； white granulated sugar （food grade）.
Instrument
Electronic balance （Shanghai Balance Instrument Factory）； biological incubator （Shanghai Biochemical Instrument Factory）； Juicer； fermenter； beater.
Methods
Acidity was determined by potentiometric titration according to GBT15038-2006； sugar content was determined by direct titration according to GBT15038-2006； and flavor and color were investigated by sensory evaluation and blood count.
Technology to Produce Mulberry-raspberry Leaven
Process
The mulberry-raspberry leaven was produced by the steps of selection， rinsing， beating， adjustment of sugar content， addition of strain， fermentation， filtration and storage. Raw material pretreatment
Healthy and high-quality raw materials were selected and cleaned， with the addition of 0.2% sodium pyrosulfite solution， and stems were removed. Then， the raw material was broken by beating[4].
Sugar content adjusting
Pectinase was added to the broken material and stored for 2 h under the condition of 25-30 ℃ and then， white granulated sugar was added to rise the sugar content to 24°.
Fermentation
The strains used for fermentation were Lactobacillu， Acetobacter and yeast[5]. During fermentation， Lactobacillu and Acetobacter were input after yeast had been put for 2 d. The duration of fermentation was 16 d at 28 ℃.
Storage
After the fermentation， the products should be filtered， and rock sugar was used to adjust the sugar content to 25°. Then， the products were stored at 15 ℃ for 3 d.
Single Factor Test
Effect of pectinase amount on juice
At first， 0， 15， 30， 45， 60， 75 and 90 mg/L pectinase were respectively added to juice at 26 ℃ for 3 h. The impact of pectinase amount on juice ratio are provided in Table 1.
As can be seen from Table 1， the juice ratio and soluble solid content increased with the pectinase content increasing. When the pectinase dosage reached 80 mg/L， the juice ratio was 66%， and the soluble solid content was 14%[5].
Effect of fermentation on leaven
Strains were inoculated at different temperatures of 26， 28， 30 and 32 ℃， respectively， and then stored for 16 d[6]. The data gained through determination of physical and chemical indicators and taste throughout fermentation are provided in Table 2.
Table 2 shows that total acid and the amount of benefited microorganisms increased with the increase of temperature， but decreased at too high temperature， which indicating the existence of strain aging and production of other secondary metabolites at such high temperature. It was found that the leaven produced at 28 ℃ had the best taste through sensory evaluation[7].
Effect of different ratios of strains on fermentation
The set four groups were provided in Table 3 according to various mass fractions of strains， and then， fermentation was performed for 16 d at 28 ℃. Sensory evaluation and total acid determination were used to select the optimal proportion[8]. Contents of total acid under various mass fractions of strains are shown in Fig 1.
From Fig 1， it was found that the content order of total acid was 4>2>3>1. The sensory evaluation results under various mass fractions of strains are provided in Table 4. Fig 1 and Table 4 indicate that the higher content of total acid does not mean better quality. No. 3 group （the mass fractions of Lactobacillus， Acetobacter and yeast was 3%， 1% and 3% ） was the optimal strain ratio[9].
Orthogonal Experiment
The orthogonal experiment was designed based on the single factor experiment.
Table 6 shows that the impact order of the three factors on the sensory evaluation of raspberry-mulberry leaven was B>C>A， which equaled temperature> strain ratio> pectinase addition. The best combination was A2B2C4， i.e.， the mass fractions of Lactobacillus， Acetobacter and yeast of 3%， 1% and 3%， respectively， under the pectinase addition of 80 mg/L and temperature at 28 or 30 ℃[10].
An extra experiment was set due to the absence of combination A2B2C4 in the orthogonal table， indicating a sensory evaluation grade at 89 points， higher than 87 points in the A2B3C4 conditions[11]. However， taking the cost of production into account， with the same quality of enzymes， low fermentation temperature is conducive to cost savings， so A2B2C4 combination should be chosen.[12].
Conclusion
Through the single factor experiment and the orthogonal experiment， the optimized fermentation condition were determined： the concentration of pectinase should be 80 mg/L， the mass fraction of Lactobacillus， Acetobacter and yeast should be 3%， 1% and 3%， respectively， the fermentation temperature should be 28 ℃， the initial sugar content should be 24°， and the duration of fermentation should be 16 d[14] . After the fermentation， the pH value was 2.6， the content of total acid in the raspberry-mulberry leaven was 61.2 g/L， and the probiotic amount was 8.5 × 108 which is in line with international standards. The product is dark red， clear and transparent， mellow and refreshing， with a unique fruit taste of mulberry and raspberry.
References
[1] LI J. Study on quality control standards of leaven drink[D]. Kunming： Kunming University of Science and Technology， 2016.
[2] GUO YP， ZHAO JA. Preliminary study on antioxidant properties of barley-ferment during spontaneous fermentation process[J]. Shanxi Chemical Industry， 2014， （04）： 4-6
[3] AI L T， GILLIAN H， JULIA C. Yeast ecology of Kombucha fermentation[J]. International Journal of Food Microbiology， 2004， 95（2）： 119-126
[4] GUO S. The studies on the health care functions of Aodong fermented beverage[J]. Jilin： Jilin University， 2015. [5] ZHANG Q， XIN XL， YANG FM， et al. Changes in antioxidant properties of red raspberry vinegar during fermentation[J]. Food Science， 2016， 37（3）：6-11
[6] KOMATSUZAKI N， TSULAHARA K， TOYOSHIMA H， et al. Effect of soaking and gaseous treatment on GABA content in germinated brownrice[J]. J Food Eng， 2007， 78（2）： 556-560.
[7] LI ZJ， NIU GC， LU BX， et al. Study on fermentation technological conditions of brown rice[J]. China Brewing， 2008， 27（22）： 65-67.
[8] HE JX. Analysis of nutritional leaven dietary supplement industry in Taiwan[J]. Technology and Industry Across the Straits， 2013（10）： 77-82.
[9] XU H， WANG QL， WEI G， et al. The health benefits and research progress of Pitaya[J]. Journal of Guangxi Academy of Sciences， 2010， 26（3）： 383-385.
[10] SAFAK S. Study on antimicrobial activities of yeasts isolated from Kombucha tea[J]. Gida， 2003， 28 （1）： 105-108.
[11] JIANG ZL， MAO JW， HUANG J， et al. Changes in antioxidant activity of grape-ferment in vitro during natural fermentation process[J]. Journal of Chinese Institute of Food Science and Technology， 2014， 14（10）： 29-34.
[12] DONG YM， HE CF， WANG L， et al. Study on the bioactivity of pitaya enzyme[J]. Food Science and Technology， 2009， 34（3）： 192-196.
[13] LYU， QI S， JIA L， et al. Study on the fermentation techniques of brown rice leaven[J]. Food Research and Development， 2007， 28（10）： 111-112.
[14] MAO JW， WU YF， FANG S. Research progress on microbial ferment food[J]. bulletin of fermentation science and technology， 2010，39（3）： 42-44.
Key words Fermentation； Mixed； Optimization
Mature mulberry contains following substances per 100 g： heat 57 kcal， dietary fiber 4.1 g， protein 1.7 g， calcium 37 mg， carbohydrate 13.8 g， iron 0.4 mg， sodium 2 mg and potassium 32 mg， but no cholesterol and saturated fat. Mulberry， considered to be a nutritious fruit for liver and kidney in Chinese medicine， is often utilized as diuretic health care food what has been recorded in Compendium of Materia Medica， a medical book in ancient China[2].
Raspberry， one of the third generation fruit popular in the world at present， has a high level of nutrition， pigment and Vitamin C and 21 unique ingredients in it[3]. Raspberry could relieve the pain due to drunk， sore throat， multiple abscesses and mastitis.
Materials and Methods
Materials
Raspberry， sold in local market； mulberry， sold in local market； pectinase， purchased from Hunan Yotel Biochemical Co.， Ltd.； white granulated sugar （food grade）.
Instrument
Electronic balance （Shanghai Balance Instrument Factory）； biological incubator （Shanghai Biochemical Instrument Factory）； Juicer； fermenter； beater.
Methods
Acidity was determined by potentiometric titration according to GBT15038-2006； sugar content was determined by direct titration according to GBT15038-2006； and flavor and color were investigated by sensory evaluation and blood count.
Technology to Produce Mulberry-raspberry Leaven
Process
The mulberry-raspberry leaven was produced by the steps of selection， rinsing， beating， adjustment of sugar content， addition of strain， fermentation， filtration and storage. Raw material pretreatment
Healthy and high-quality raw materials were selected and cleaned， with the addition of 0.2% sodium pyrosulfite solution， and stems were removed. Then， the raw material was broken by beating[4].
Sugar content adjusting
Pectinase was added to the broken material and stored for 2 h under the condition of 25-30 ℃ and then， white granulated sugar was added to rise the sugar content to 24°.
Fermentation
The strains used for fermentation were Lactobacillu， Acetobacter and yeast[5]. During fermentation， Lactobacillu and Acetobacter were input after yeast had been put for 2 d. The duration of fermentation was 16 d at 28 ℃.
Storage
After the fermentation， the products should be filtered， and rock sugar was used to adjust the sugar content to 25°. Then， the products were stored at 15 ℃ for 3 d.
Single Factor Test
Effect of pectinase amount on juice
At first， 0， 15， 30， 45， 60， 75 and 90 mg/L pectinase were respectively added to juice at 26 ℃ for 3 h. The impact of pectinase amount on juice ratio are provided in Table 1.
As can be seen from Table 1， the juice ratio and soluble solid content increased with the pectinase content increasing. When the pectinase dosage reached 80 mg/L， the juice ratio was 66%， and the soluble solid content was 14%[5].
Effect of fermentation on leaven
Strains were inoculated at different temperatures of 26， 28， 30 and 32 ℃， respectively， and then stored for 16 d[6]. The data gained through determination of physical and chemical indicators and taste throughout fermentation are provided in Table 2.
Table 2 shows that total acid and the amount of benefited microorganisms increased with the increase of temperature， but decreased at too high temperature， which indicating the existence of strain aging and production of other secondary metabolites at such high temperature. It was found that the leaven produced at 28 ℃ had the best taste through sensory evaluation[7].
Effect of different ratios of strains on fermentation
The set four groups were provided in Table 3 according to various mass fractions of strains， and then， fermentation was performed for 16 d at 28 ℃. Sensory evaluation and total acid determination were used to select the optimal proportion[8]. Contents of total acid under various mass fractions of strains are shown in Fig 1.
From Fig 1， it was found that the content order of total acid was 4>2>3>1. The sensory evaluation results under various mass fractions of strains are provided in Table 4. Fig 1 and Table 4 indicate that the higher content of total acid does not mean better quality. No. 3 group （the mass fractions of Lactobacillus， Acetobacter and yeast was 3%， 1% and 3% ） was the optimal strain ratio[9].
Orthogonal Experiment
The orthogonal experiment was designed based on the single factor experiment.
Table 6 shows that the impact order of the three factors on the sensory evaluation of raspberry-mulberry leaven was B>C>A， which equaled temperature> strain ratio> pectinase addition. The best combination was A2B2C4， i.e.， the mass fractions of Lactobacillus， Acetobacter and yeast of 3%， 1% and 3%， respectively， under the pectinase addition of 80 mg/L and temperature at 28 or 30 ℃[10].
An extra experiment was set due to the absence of combination A2B2C4 in the orthogonal table， indicating a sensory evaluation grade at 89 points， higher than 87 points in the A2B3C4 conditions[11]. However， taking the cost of production into account， with the same quality of enzymes， low fermentation temperature is conducive to cost savings， so A2B2C4 combination should be chosen.[12].
Conclusion
Through the single factor experiment and the orthogonal experiment， the optimized fermentation condition were determined： the concentration of pectinase should be 80 mg/L， the mass fraction of Lactobacillus， Acetobacter and yeast should be 3%， 1% and 3%， respectively， the fermentation temperature should be 28 ℃， the initial sugar content should be 24°， and the duration of fermentation should be 16 d[14] . After the fermentation， the pH value was 2.6， the content of total acid in the raspberry-mulberry leaven was 61.2 g/L， and the probiotic amount was 8.5 × 108 which is in line with international standards. The product is dark red， clear and transparent， mellow and refreshing， with a unique fruit taste of mulberry and raspberry.
References
[1] LI J. Study on quality control standards of leaven drink[D]. Kunming： Kunming University of Science and Technology， 2016.
[2] GUO YP， ZHAO JA. Preliminary study on antioxidant properties of barley-ferment during spontaneous fermentation process[J]. Shanxi Chemical Industry， 2014， （04）： 4-6
[3] AI L T， GILLIAN H， JULIA C. Yeast ecology of Kombucha fermentation[J]. International Journal of Food Microbiology， 2004， 95（2）： 119-126
[4] GUO S. The studies on the health care functions of Aodong fermented beverage[J]. Jilin： Jilin University， 2015. [5] ZHANG Q， XIN XL， YANG FM， et al. Changes in antioxidant properties of red raspberry vinegar during fermentation[J]. Food Science， 2016， 37（3）：6-11
[6] KOMATSUZAKI N， TSULAHARA K， TOYOSHIMA H， et al. Effect of soaking and gaseous treatment on GABA content in germinated brownrice[J]. J Food Eng， 2007， 78（2）： 556-560.
[7] LI ZJ， NIU GC， LU BX， et al. Study on fermentation technological conditions of brown rice[J]. China Brewing， 2008， 27（22）： 65-67.
[8] HE JX. Analysis of nutritional leaven dietary supplement industry in Taiwan[J]. Technology and Industry Across the Straits， 2013（10）： 77-82.
[9] XU H， WANG QL， WEI G， et al. The health benefits and research progress of Pitaya[J]. Journal of Guangxi Academy of Sciences， 2010， 26（3）： 383-385.
[10] SAFAK S. Study on antimicrobial activities of yeasts isolated from Kombucha tea[J]. Gida， 2003， 28 （1）： 105-108.
[11] JIANG ZL， MAO JW， HUANG J， et al. Changes in antioxidant activity of grape-ferment in vitro during natural fermentation process[J]. Journal of Chinese Institute of Food Science and Technology， 2014， 14（10）： 29-34.
[12] DONG YM， HE CF， WANG L， et al. Study on the bioactivity of pitaya enzyme[J]. Food Science and Technology， 2009， 34（3）： 192-196.
[13] LYU， QI S， JIA L， et al. Study on the fermentation techniques of brown rice leaven[J]. Food Research and Development， 2007， 28（10）： 111-112.
[14] MAO JW， WU YF， FANG S. Research progress on microbial ferment food[J]. bulletin of fermentation science and technology， 2010，39（3）： 42-44.